U.S. patent application number 13/458927 was filed with the patent office on 2012-11-29 for tolerogenic synthetic nanocarriers coupled to cd1d-restricted antigens and methods of use.
This patent application is currently assigned to Selecta Biosciences, Inc.. Invention is credited to Christopher Fraser, Takashi Kei Kishimoto, Roberto A. Maldonado.
Application Number | 20120301510 13/458927 |
Document ID | / |
Family ID | 47068065 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301510 |
Kind Code |
A1 |
Kishimoto; Takashi Kei ; et
al. |
November 29, 2012 |
TOLEROGENIC SYNTHETIC NANOCARRIERS COUPLED TO CD1D-RESTRICTED
ANTIGENS AND METHODS OF USE
Abstract
Disclosed are synthetic nanocarrier compositions, and related
methods, comprising CD1d-restricted antigens and immunosuppressants
that provide tolerogenic immune responses.
Inventors: |
Kishimoto; Takashi Kei;
(Lexington, MA) ; Fraser; Christopher; (Arlington,
MA) ; Maldonado; Roberto A.; (Jamaica Plain,
MA) |
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
47068065 |
Appl. No.: |
13/458927 |
Filed: |
April 27, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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61480946 |
Apr 29, 2011 |
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61513514 |
Jul 29, 2011 |
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61531147 |
Sep 6, 2011 |
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61531153 |
Sep 6, 2011 |
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61531164 |
Sep 6, 2011 |
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61531168 |
Sep 6, 2011 |
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61531175 |
Sep 6, 2011 |
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61531180 |
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61531194 |
Sep 6, 2011 |
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61531204 |
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Current U.S.
Class: |
424/400 ;
424/193.1; 424/197.11; 977/735; 977/754; 977/762; 977/773; 977/788;
977/906 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/13 20130101; A61K 9/127 20130101; A61P 35/00 20180101; G01N
33/56972 20130101; G01N 2333/70514 20130101; A61K 9/51 20130101;
A61K 39/00 20130101; A61P 37/02 20180101; A61P 37/06 20180101; A61K
39/385 20130101; A61P 11/02 20180101; A61K 47/593 20170801; A61P
37/04 20180101; B82Y 5/00 20130101; A61P 11/06 20180101; A61K 9/14
20130101; A61K 47/643 20170801; A61P 41/00 20180101; A61K 47/6937
20170801; A61P 7/06 20180101; A61P 1/16 20180101; G01N 2333/7051
20130101; A61K 39/0008 20130101; A61K 47/69 20170801; A61K 47/6929
20170801; A61K 2039/577 20130101; A61P 37/00 20180101; A61K
2039/55555 20130101; A61K 38/38 20130101; A61P 17/00 20180101; A61P
37/08 20180101; A61K 39/35 20130101; A61K 39/36 20130101; A61K
47/544 20170801; B82Y 40/00 20130101; A61K 47/50 20170801; G01N
33/505 20130101; A61P 29/00 20180101; A61K 47/52 20170801; A61P
15/00 20180101; Y02A 50/30 20180101; A61K 2039/55511 20130101; G01N
2333/70517 20130101; A61K 31/192 20130101; A61K 38/1816 20130101;
A61K 9/5146 20130101; A61K 39/001 20130101; A61K 9/5153 20130101;
A61K 47/6923 20170801; A61K 9/5115 20130101; A61K 2039/5154
20130101; A61K 39/36 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/400 ;
424/193.1; 424/197.11; 977/788; 977/773; 977/754; 977/735; 977/762;
977/906 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61P 37/06 20060101 A61P037/06; A61P 37/08 20060101
A61P037/08; A61P 1/16 20060101 A61P001/16; A61P 7/06 20060101
A61P007/06; A61P 15/00 20060101 A61P015/00; A61K 9/14 20060101
A61K009/14; A61P 29/00 20060101 A61P029/00 |
Claims
1. A composition comprising: (i) a first population of synthetic
nanocarriers coupled to immunosuppressants, and (ii) a second
population of synthetic nanocarriers coupled to CD1d-restricted
antigens.
2. The composition of claim 1, wherein the first population and
second population are the same population.
3. The composition of claim 1, wherein the immunosuppressants
comprise a statin, an mTOR inhibitor, a TGF-.beta. signaling agent,
a corticosteroid, an inhibitor of mitochondrial function, a P38
inhibitor, an NF-.kappa..beta. inhibitor, an adenosine receptor
agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4
inhibitor, an HDAC inhibitor or a proteasome inhibitor.
4. (canceled)
5. The composition of claim 1, wherein the CD1d-restricted antigens
comprise glycolipids.
6. The composition of claim 1, wherein the CD1d-restricted antigens
comprise .alpha.-galactosylceramide, .beta.-glucosylceramide,
.alpha.-linked glycosphingolipid from Sphingomonas spp., galactosyl
diaglycerol from Borrelia burgdorferi, lypophosphoglycan from
Leishmania or phosphatidylinositol tetramannoside from
Mycobacterium leprae.
7. The composition of claim 1, wherein the composition is in an
amount effective to generate a tolerogenic immune response when
administered to a subject.
8-9. (canceled)
10. The composition of claim 1, wherein the composition further
comprises APC presentable antigens.
11-13. (canceled)
14. The composition of claim 10, wherein the APC presentable
antigens are autoantigens, allergens, or are associated with an
inflammatory disease, fatty liver disease, an autoimmune disease,
sickle cell disease, spontaneous abortion organ or tissue rejection
or graft versus host disease.
15. The composition of claim 1, wherein the load of the
immunosuppressants and/or CD1d-restricted antigens on average
across the first and/or second population of synthetic nanocarriers
is between 0.0001% and 50%.
16. (canceled)
17. The composition of claim 1, wherein the synthetic nanocarriers
of the first population and/or second population and/or third
population comprise lipid nanoparticles, polymeric nanoparticles,
metallic nanoparticles, surfactant-based emulsions, dendrimers,
buckyballs, nanowires, virus-like particles or peptide or protein
particles.
18-27. (canceled)
28. The composition of claim 1, wherein the mean of a particle size
distribution obtained using dynamic light scattering of the
synthetic nanocarriers of the first and/or second and/or third
population is a diameter greater than 100 nm.
29-32. (canceled)
33. The composition of claim 1, wherein the aspect ratio of the
synthetic nanocarriers of the first population and/or second
population and/or third population is greater than 1:1, 1:1.2,
1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.
34. (canceled)
35. A dosage form comprising the composition of claim 1.
36. A method comprising administering the composition of claim 1 to
a subject.
37. A method comprising: administering to a subject a composition
comprising: (i) a first population of synthetic nanocarriers
coupled to immunosuppressants, and (ii) a second population of
synthetic nanocarriers coupled to CD1d-restricted antigens, wherein
the composition is in an amount effective to generate a tolerogenic
immune response in the subject.
38. A method comprising: generating a tolerogenic immune response
in a subject by administering a composition comprising: (i) a first
population of synthetic nanocarriers coupled to immunosuppressants,
and (ii) a second population of synthetic nanocarriers coupled to
CD1d-restricted antigens.
39. A method comprising: administering a composition to a subject
according to a protocol that was previously shown to generate a
tolerogenic immune response in one or more test subjects; wherein
the composition comprises: (i) a first population of synthetic
nanocarriers coupled to immunosuppressants, and (ii) a second
population of synthetic nanocarriers coupled to CD1d-restricted
antigens.
40-81. (canceled)
82. A method comprising: (i) producing a first population of
synthetic nanocarriers coupled to immunosuppressants, and (ii)
producing a second population of synthetic nanocarriers coupled to
CD1d-restricted antigens.
83-93. (canceled)
94. A process for producing a composition or dosage form comprising
the steps of: (i) coupling a first population of synthetic
nanocarriers to immunosuppressants, and (ii) coupling a second
population of synthetic nanocarriers to CD1d-restricted
antigens.
95. (canceled)
96. A composition or dosage form obtainable by the method of claim
82.
97-100. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119 of U.S. provisional application 61/480,946, filed Apr.
29, 2011, 61/513,514, filed Jul. 29, 2011, 61/531,147, filed Sep.
6, 2011, 61/531,153, filed Sep. 6, 2011, 61/531,164, filed Sep. 6,
2011, 61/531,168, filed Sep. 6, 2011, 61/531,175, filed Sep. 6,
2011, 61/531,180, filed Sep. 6, 2011, 61/531,194, filed Sep. 6,
2011, 61/531,204, filed Sep. 6, 2011, 61/531,209, filed Sep. 6,
2011, 61/531,215, filed Sep. 6, 2011, the entire contents of each
of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic nanocarrier compositions
with CD1d-restricted antigens and immunosuppressants, and related
methods. The compositions and methods allow for the stimulation of
tolerogenic immune responses in a subject via iNKT cells. The
tolerogenic immune responses can be antigen-specific, and the
compositions provided may also include APC presentable antigens.
Such compositions allow for efficient uptake by APCs to shift the
immune response in favor of tolerogenic immune response
development.
BACKGROUND OF THE INVENTION
[0003] Conventional immunosuppressant drugs are broad-acting.
Additionally, in order to maintain immunosuppression,
immunosuppressant drug therapy is generally a life-long
proposition. Unfortunately, the use of broad-acting
immunosuppressants are associated with a risk of severe side
effects, such as tumors, infections, nephrotoxicity and metabolic
disorders. Accordingly, new immunosuppressant therapies would be
beneficial.
SUMMARY OF THE INVENTION
[0004] In one aspect, a composition comprising (i) a first
population of synthetic nanocarriers coupled to immunosuppressants,
and (ii) a second population of synthetic nanocarriers coupled to
CD1d-restricted antigens is provided. In one embodiment, the first
population and second population are the same population. In
another embodiment, the first population and second population are
different populations.
[0005] In another embodiment, the immunosuppressants comprise a
statin, an mTOR inhibitor, a TGF-.beta. signaling agent, a
corticosteroid, an inhibitor of mitochondrial function, a P38
inhibitor, an NF-.kappa..beta. inhibitor, an adenosine receptor
agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4
inhibitor, an HDAC inhibitor or a proteasome inhibitor. In another
embodiment, the mTOR inhibitor is rapamycin or a rapamycin
analog.
[0006] In another embodiment, the CD1d-restricted antigens comprise
glycolipids. In another embodiment, the CD1d-restricted antigens
comprise .alpha.-galactosylceramide, .beta.-glucosylceramide,
.alpha.-linked glycosphingolipid from Sphingomonas spp., galactosyl
diaglycerol from Borrelia burgdorferi, lypophosphoglycan from
Leishmania or phosphatidylinositol tetramannoside from
Mycobacterium leprae.
[0007] In another embodiment, the composition is in an amount
effective to generate a tolerogenic immune response when
administered to a subject. In one embodiment, the tolerogenic
immune response is the stimulation of iNKT cells. In another
embodiment, the tolerogenic immune response is the production of
IL-4 by iNKT cells. In another embodiment, the tolerogenic immune
response is the production of IL-10 by iNKT cells. In another
embodiment, the tolerogenic immune response is iNKT cell
stimulation. In another embodiment, the tolerogenic immune response
is iNKT cell anergy or the reduction in pathogenic iNKT cells. In
another embodiment, the tolerogenic immune response is a reduction
in IFN-.gamma. produced by iNKT cells. In another embodiment, the
tolerogenic immune response is antigen-specific.
[0008] In another embodiment, the composition further comprises APC
presentable antigens. In another embodiment, the APC presentable
antigens are coupled to the first and/or second populations of
synthetic nanocarriers. In another embodiment, the APC presentable
antigens are coupled to a third population of synthetic
nanocarriers. In one embodiment, the coupling is covalent coupling.
In another embodiment, the coupling is noncovalent coupling. In
another embodiment, the APC presentable antigens are not coupled to
synthetic nanocarriers.
[0009] In another embodiment, the APC presentable antigens comprise
MHC Class I-restricted, MHC Class II-restricted epitopes and/or B
cell epitopes. In yet another embodiment, the APC presentable
antigens comprise substantially no B cell epitopes. In one
embodiment, the APC presentable antigens are or are obtained or
derived from proteins, polypeptides, polysaccharides,
polynucleotides or cells.
[0010] In a further embodiment, the composition further comprises a
transplantable graft.
[0011] In another embodiment, the APC presentable antigens are
autoantigens, allergens, or are associated with an inflammatory
disease, fatty liver disease, an autoimmune disease, allergy,
sickle cell disease, spontaneous abortion organ or tissue rejection
or graft versus host disease.
[0012] In another embodiment, the load of the immunosuppressants
and/or CD1d-restricted antigens on average across the first and/or
second population of synthetic nanocarriers is between 0.0001% and
50%. In another embodiment, the load of the immunosuppressants
and/or CD1d-restricted antigens on average across the first and/or
second population of synthetic nanocarriers is between 0.1% and
10%.
[0013] In another embodiment, the synthetic nanocarriers of the
first population and/or second population and/or third population
comprise lipid nanoparticles, polymeric nanoparticles, metallic
nanoparticles, surfactant-based emulsions, dendrimers, buckyballs,
nanowires, virus-like particles or peptide or protein particles. In
another embodiment, the synthetic nanocarriers of the first and/or
second and/or third populations comprise lipid nanoparticles. In
another embodiment, the synthetic nanocarriers of the first and/or
second and/or third populations comprise liposomes. In another
embodiment, the synthetic nanocarriers of the first and/or second
and/or third populations comprise metallic nanoparticles. In
another embodiment, the metallic nanoparticles comprise gold
nanoparticles. In another embodiment, the synthetic nanocarriers of
the first and/or second and/or third populations comprise polymeric
nanoparticles. In another embodiment, the polymeric nanoparticles
comprise polymer that is a non-methoxy-terminated, pluronic
polymer. In another embodiment, the polymeric nanoparticles
comprise a polyester, a polyester coupled to a polyether, polyamino
acid, polycarbonate, polyacetal, polyketal, polysaccharide,
polyethyloxazoline or polyethyleneimine. In another embodiment, the
polyester comprises a poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid) or polycaprolactone. In another
embodiment, the polymeric nanoparticles comprise a polyester and a
polyester coupled to a polyether. In another embodiment, the
polyether comprises polyethylene glycol or polypropylene
glycol.
[0014] In another embodiment, the mean of a particle size
distribution obtained using dynamic light scattering of the
synthetic nanocarriers of the first and/or second and/or third
population is a diameter greater than 100 nm. In another
embodiment, the diameter is greater than 150 nm. In another
embodiment, the diameter is greater than 200 nm. In another
embodiment, the diameter is greater than 250 nm. In another
embodiment, the diameter is greater than 300 nm.
[0015] In another embodiment, the aspect ratio of the synthetic
nanocarriers of the first population and/or second population
and/or third population is greater than 1:1, 1:1.2, 1:1.5, 1:2,
1:3, 1:5, 1:7 or 1:10.
[0016] In another embodiment, the composition further comprises a
pharmaceutically acceptable excipient.
[0017] In another aspect, a dosage form comprising any of the
compositions provided herein is provided.
[0018] In another aspect, a method comprising administering any of
the compositions or dosage forms to a subject is provided.
[0019] In another aspect, a method comprising administering to a
subject a composition comprising (i) a first population of
synthetic nanocarriers coupled to immunosuppressants, and (ii) a
second population of synthetic nanocarriers coupled to
CD1d-restricted antigens, wherein the composition is in an amount
effective to generate a tolerogenic immune response in the subject
is provided. In another aspect, a method comprising generating a
tolerogenic immune response in a subject by administering a
composition comprising (i) a first population of synthetic
nanocarriers coupled to immunosuppressants, and (ii) a second
population of synthetic nanocarriers coupled to CD1d-restricted
antigens is provided. In another aspect, a method comprising
administering a composition to a subject according to a protocol
that was previously shown to generate a tolerogenic immune response
in one or more test subjects; wherein the composition comprises (i)
a first population of synthetic nanocarriers coupled to
immunosuppressants, and (ii) a second population of synthetic
nanocarriers coupled to CD1d-restricted antigens is provided
[0020] In one embodiment, the first population and second
population are the same population. In another embodiment, the
first population and second population are different
populations.
[0021] In another embodiment, the method further comprises
providing or identifying the subject.
[0022] In another embodiment, the immunosuppressants comprise a
statin, an mTOR inhibitor, a TGF-.beta. signaling agent, a
corticosteroid, an inhibitor of mitochondrial function, a P38
inhibitor, an NF-.kappa..beta. inhibitor, an adenosine receptor
agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4
inhibitor, an HDAC inhibitor or a proteasome inhibitor. In another
embodiment, the mTOR inhibitor is rapamycin or a rapamycin
analog.
[0023] In another embodiment, the CD1d-restricted antigens comprise
glycolipids. In another embodiment, the CD1d-restricted antigens
comprise .alpha.-galactosylceramide, .beta.-glucosylceramide,
.alpha.-linked glycosphingolipid from Sphingomonas spp., galactosyl
diaglycerol from Borrelia burgdorferi, lypophosphoglycan from
Leishmania or phosphatidylinositol tetramannoside from
Mycobacterium leprae.
[0024] In another embodiment, a tolerogenic immune response is
generated in the subject. In one embodiment, the tolerogenic immune
response is the stimulation of iNKT cells. In another embodiment,
the tolerogenic immune response is the production of IL-4 by iNKT
cells. In another embodiment, the tolerogenic immune response is
the production of IL-10 by iNKT cells. In another embodiment, the
tolerogenic immune response is iNKT cell stimulation. In another
embodiment, the tolerogenic immune response is iNKT cell anergy or
the reduction in pathogenic iNKT cells. In another embodiment, the
tolerogenic immune response is a reduction in IFN-.gamma. produced
by iNKT cells. In another embodiment, the tolerogenic immune
response is antigen-specific.
[0025] In another embodiment, the method further comprises
administering APC presentable antigens to the subject. In one
embodiment, the APC presentable antigens are administered prior to,
concomitantly with or after the administration of the composition
comprising the first population and second population of synthetic
nanocarriers. In another embodiment, the APC presentable antigens
are coupled to the first and/or second populations of synthetic
nanocarriers. In another embodiment, the APC presentable antigens
are coupled to a third population of synthetic nanocarriers. In one
embodiment, the coupling is covalent coupling. In another
embodiment, the coupling is noncovalent coupling. In another
embodiment, the APC presentable antigens are not coupled to
synthetic nanocarriers.
[0026] In another embodiment, the APC presentable antigens comprise
MHC Class I-restricted, MHC Class II-restricted epitopes and/or B
cell epitopes. In yet another embodiment, the APC presentable
antigens comprise substantially no B cell epitopes. In one
embodiment, the APC presentable antigens are or are obtained or
derived from proteins, polypeptides, polysaccharides,
polynucleotides or cells.
[0027] In a further embodiment, the method further comprises
administering a transplantable graft.
[0028] In another embodiment, the APC presentable antigens are
autoantigens, allergens, or are associated with an inflammatory
disease, fatty liver disease, an autoimmune disease, an allergy,
sickle cell disease, spontaneous abortion organ or tissue rejection
or graft versus host disease.
[0029] In another embodiment, the load of the immunosuppressants
and/or CD1d-restricted antigens on average across the first and/or
second population of synthetic nanocarriers is between 0.0001% and
50%. In another embodiment, the load of the immunosuppressants
and/or CD1d-restricted antigens on average across the first and/or
second population of synthetic nanocarriers is between 0.1% and
10%.
[0030] In another embodiment, the synthetic nanocarriers of the
first population and/or second population and/or third population
comprise lipid nanoparticles, polymeric nanoparticles, metallic
nanoparticles, surfactant-based emulsions, dendrimers, buckyballs,
nanowires, virus-like particles or peptide or protein particles. In
another embodiment, the synthetic nanocarriers of the first and/or
second and/or third populations comprise lipid nanoparticles. In
another embodiment, the synthetic nanocarriers of the first and/or
second and/or third populations comprise liposomes. In another
embodiment, the synthetic nanocarriers of the first and/or second
and/or third populations comprise metallic nanoparticles. In
another embodiment, the metallic nanoparticles comprise gold
nanoparticles. In another embodiment, the synthetic nanocarriers of
the first and/or second and/or third populations comprise polymeric
nanoparticles. In another embodiment, the polymeric nanoparticles
comprise polymer that is a non-methoxy-terminated, pluronic
polymer. In another embodiment, the polymeric nanoparticles
comprise a polyester, a polyester coupled to a polyether, polyamino
acid, polycarbonate, polyacetal, polyketal, polysaccharide,
polyethyloxazoline or polyethyleneimine. In another embodiment, the
polyester comprises a poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid) or polycaprolactone. In another
embodiment, the polymeric nanoparticles comprise a polyester and a
polyester coupled to a polyether. In another embodiment, the
polyether comprises polyethylene glycol or polypropylene
glycol.
[0031] In another embodiment, the mean of a particle size
distribution obtained using dynamic light scattering of the
synthetic nanocarriers of the first and/or second and/or third
population is a diameter greater than 100 nm. In another
embodiment, the diameter is greater than 150 nm. In another
embodiment, the diameter is greater than 200 nm. In another
embodiment, the diameter is greater than 250 nm. In another
embodiment, the diameter is greater than 300 nm.
[0032] In another embodiment, the aspect ratio of the synthetic
nanocarriers of the first population and/or second population
and/or third population is greater than 1:1, 1:1.2, 1:1.5, 1:2,
1:3, 1:5, 1:7 or 1:10.
[0033] In another embodiment, one or more maintenance doses of the
composition comprising the first population and second population
of synthetic nanocarriers and/or the APC presentable antigens are
administered to the subject.
[0034] In another embodiment, the method further comprises
assessing the generation of a/the tolerogenic immune response in
the subject prior to and/or after the administration of the
composition comprising the first population and second population
of synthetic nanocarriers and/or the APC presentable antigens. In
one embodiment, the tolerogenic immune response is the stimulation
of iNKT cells. In another embodiment, the tolerogenic immune
response is the production of IL-4 by iNKT cells. In another
embodiment, the tolerogenic immune response is the production of
IL-10 by iNKT cells. In another embodiment, the tolerogenic immune
response is iNKT cell stimulation. In another embodiment, the
tolerogenic immune response is iNKT cell anergy or the reduction in
pathogenic iNKT cells. In another embodiment, the tolerogenic
immune response is a reduction in IFN-.gamma. produced by iNKT
cells. In another embodiment, the tolerogenic immune response is
antigen-specific.
[0035] In another embodiment, the subject has or is at risk of
having an inflammatory disease, fatty liver disease, an autoimmune
disease, sickle cell disease, an allergy, spontaneous abortion,
organ or tissue rejection or graft versus host disease. In another
embodiment, the subject has undergone or will undergo
transplantation.
[0036] In another embodiment, the administering of the synthetic
nanocarriers and/or APC presentable antigens is by intravenous,
intraperitoneal, transmucosal, oral, subcutaneous, pulmonary,
intranasal, intradermal or intramuscular administration. In another
embodiment, the administering of the synthetic nanocarriers and/or
APC presentable antigens is by inhalation or intravenous,
subcutaneous or transmucosal administration.
[0037] In another aspect, a method comprising (i) producing a first
population of synthetic nanocarriers coupled to immunosuppressants,
and (ii) producing a second population of synthetic nanocarriers
coupled to CD1d-restricted antigens is provided.
[0038] In one embodiment, the first population and second
population are the same population. In another embodiment, the
first population and second population are different
populations.
[0039] In another embodiment, the method further comprises
including APC presentable antigens with the first population and/or
second population of the synthetic nanocarriers. In one embodiment,
the APC presentable antigens are coupled to the first population
and/or second population of synthetic nanocarriers. In another
embodiment, the APC presentable antigens are coupled to a third
population of synthetic nanocarriers. In another embodiment, the
coupling is covalent coupling. In another embodiment, the coupling
is non-covalent coupling. In another embodiment, the APC
presentable antigens are not coupled to synthetic nanocarriers.
[0040] In another embodiment, the first population and second
population and/or third population of synthetic nanocarriers that
are produced are as defined in any of the compositions or methods
provided herein. In another embodiment, the method further
comprises producing a dosage form of a composition comprising the
first population and second population and/or third population of
synthetic nanocarriers produced. In another embodiment, the method
further comprises making a composition comprising the first
population and second population of synthetic nanocarriers and/or
third population of synthetic nanocarriers or the dosage form
available to a subject for administration. In another embodiment,
the method further comprises assessing the generation of a
tolerogenic immune response with a composition comprising the first
population and second population of synthetic nanocarriers and/or
third population of synthetic nanocarriers or the dosage form.
[0041] In one embodiment, the tolerogenic immune response is the
stimulation of iNKT cells. In another embodiment, the tolerogenic
immune response is the production of IL-4 by iNKT cells. In another
embodiment, the tolerogenic immune response is the production of
IL-10 by iNKT cells. In another embodiment, the tolerogenic immune
response is iNKT cell stimulation. In another embodiment, the
tolerogenic immune response is iNKT cell anergy or the reduction in
pathogenic iNKT cells. In another embodiment, the tolerogenic
immune response is a reduction in IFN-.gamma. produced by iNKT
cells. In another embodiment, the tolerogenic immune response is
antigen-specific.
[0042] In another aspect, a process for producing a composition or
dosage form comprising the steps of (i) coupling a first population
of synthetic nanocarriers to immunosuppressants, and (ii) coupling
a second population of synthetic nanocarriers to CD1d-restricted
antigens is provided. In one embodiment, the process comprises the
steps as defined in any of the methods provided herein.
[0043] In another aspect, a composition or dosage form obtainable
by any of the methods or processes provided herein is provided.
[0044] In another aspect, any of the compositions or dosage forms
provided may be for use in therapy or prophylaxis.
[0045] In another aspect, any of the compositions or dosage forms
provided may be for use in a method of generating a tolerogenic
immune response in a subject, the treatment or prophylaxis of
allergy, sickle cell disease, autoimmune disease, inflammatory
disease, fatty liver disease, spontaneous abortion, organ or tissue
rejection or graft versus host disease or in any of the methods
provided herein.
[0046] In another aspect, a use of any of the compositions or
dosage forms provided for the manufacture of a medicament for use
in a method of generating a tolerogenic immune response in a
subject, the treatment or prophylaxis of allergy, sickle cell
disease, autoimmune disease, inflammatory disease, fatty liver
disease, spontaneous abortion, organ or tissue rejection or graft
versus host disease or in any of the methods provided herein is
provided.
[0047] In another aspect, a dosage form comprising any of the
compositions provided herein is provided.
[0048] In another embodiment, of any of the compositions and
methods provided herein, the CD1d-restricted antigen comprises
substantially no B cell epitopes.
BRIEF DESCRIPTION OF FIGURES
[0049] FIG. 1 provides a representative example of a flow
cytometric analysis of Treg cells.
[0050] FIG. 2 shows that synthetic nanocarriers comprising
immunosuppressant reduces the level of CD69 expression, a marker
for T cell activation.
[0051] FIG. 3 shows the level of IFN-.gamma. expression by iNKT
cells.
[0052] FIG. 4 shows the level of IL-4 expression by iNKT cells.
[0053] FIG. 5 shows the level of IL-10 expression by iNKT
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
[0055] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes. As used in this
specification and the appended claims, the singular forms "a," "an"
and "the" include plural referents unless the content clearly
dictates otherwise. For example, reference to "a polymer" includes
a mixture of two or more such molecules or a mixture of differing
molecular weights of a single polymer species, reference to "a
synthetic nanocarrier" includes a mixture of two or more such
synthetic nanocarriers or a plurality of such synthetic
nanocarriers, reference to "a DNA molecule" includes a mixture of
two or more such DNA molecules or a plurality of such DNA
molecules, reference to "an immunosuppressant" includes mixture of
two or more such materials or a plurality of immunosuppressant
molecules, and the like.
[0056] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited integer (e.g. a feature, element,
characteristic, property, method/process step or limitation) or
group of integers (e.g. features, element, characteristics,
properties, method/process steps or limitations) but not the
exclusion of any other integer or group of integers. Thus, as used
herein, the term "comprising" is inclusive and does not exclude
additional, unrecited integers or method/process steps.
[0057] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of". The phrase "consisting
essentially of" is used herein to require the specified integer(s)
or steps as well as those which do not materially affect the
character or function of the claimed invention. As used herein, the
term "consisting" is used to indicate the presence of the recited
integer (e.g. a feature, element, characteristic, property,
method/process step or limitation) or group of integers (e.g.
features, element, characteristics, properties, method/process
steps or limitations) alone.
A. INTRODUCTION
[0058] NKT cells are a heterogeneous group of T cells that can be
divided into invariant NKT cells and non-invariant NKT cells.
Invariant NKT (iNKT) cells are a population of innate T cells that
are restricted by the non-classical MHC Class I molecule, CD1d.
CD1d is an antigen-presenting molecule that binds self- and foreign
lipids and glycolipids and is often found on antigen-presenting
cells, but also on non-hematopoietic cells such as hepatocytes.
Upon stimulation, iNKT cells, in some embodiments, are able to
enhance tolerogenic effects of the immune system, such as by
shifting to the production of anti-inflammatory cytokines (e.g.,
IL-10 and IL-4). As illustrated in the Examples below, it has been
found that synthetic nanocarriers that comprise immunosuppressant
and CD1d-restricted antigens are able to elicit such tolerogenic
effects. It has been shown that such synthetic nanocarriers were
effective in producing the aforementioned cytokines when contacted
with the nanocarriers. In some embodiments, the compositions
provided herein can result in iNKT cell anergy where their
activation and production of cytokines is reduced (e.g.,
inflammatory cytokines, such as IFN-.gamma.). In some embodiments,
such as where iNKT cell activity is pathogenic (e.g., asthma) iNKT
cell anergy or a decrease in cytokines, such as IL-4, may be
beneficial. In other embodiments, the compositions provided herein
can result in iNKT cell stimulation. With the compositions and
methods provided, it is possible to cause iNKT cells to promote
tolerogenic effects, which have application in a number of disease
contexts. The compositions provided herein are also expected to be
delivered more directly at the sites of action in cells of
interest, in particular APCs, and can not only result in beneficial
tolerogenic immune responses but also a reduction in off-target
effects and toxicity. This invention is useful for promoting
tolerogenic immune responses in a variety of subjects, such as
those that have or are at risk of having an inflammatory disease,
an autoimmune disease, sickle cell disease, fatty liver disease,
spontaneous abortion, an allergy, organ or tissue rejection or
graft verus host disease. Such subjects also include those who have
undergone or will undergo transplantation.
[0059] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, the
inventors have unexpectedly discovered that it is possible to
provide synthetic nanocarrier compositions, and related methods,
that induce a tolerogenic immune response. The compositions
described herein include compositions that comprise (i) a first
population of synthetic nanocarriers coupled to immunosuppressants,
and (ii) a second population of synthetic nanocarriers coupled to
CD1d-restricted antigens. In embodiments, the compositions further
comprise an APC presentable antigen that may or may not be coupled
to the first or second population of synthetic nanocarriers or
another population of synthetic nanocarriers. The presence of the
APC presentable antigens can result in further antigen-specific
tolerogenic immune responses.
[0060] In another aspect, dosage forms of any of the compositions
herein are provided. Such dosage forms can be administered to a
subject in need thereof (e.g., in need of tolerogenic immune
responses).
[0061] In another aspect, any of the compositions provided herein
is administered to a subject. The composition may be administered
in an amount effective to generate a tolerogenic immune response in
the subject. In one embodiment, a composition is administered to a
subject according to a protocol that was previously shown to
generate a tolerogenic immune response in one or more subjects.
[0062] Other antigens, such as APC presentable antigens, may also
be administered to the subject provided herein. Such antigens may
be administered to a subject prior to, concomitantly with or after
the administration of the first and second populations of synthetic
nanocarriers. Such antigens may or may not be coupled to the first
or second population of synthetic nanocarriers or another
population of synthetic nanocarriers.
[0063] In embodiments, the compositions provided may also be
administered as one or more maintenance doses to a subject. In such
embodiments, the compositions provided are administered such that
the generation of an undesired immune response is reduced or a
desired immune response is produced for a certain length of time.
Examples of such lengths of time are provided elsewhere herein.
[0064] In yet another aspect, a method of (i) producing a first
population of synthetic nanocarriers coupled to immunosuppressants,
and (ii) producing a second population of synthetic nanocarriers
coupled to CD1d-restricted antigens is provided.
[0065] The invention will now be described in more detail
below.
B. DEFINITIONS
[0066] "Administering" or "administration" means providing a
material to a subject in a manner that is pharmacologically
useful.
[0067] "Allergens" are any substances that can cause an undesired
(e.g., a Type 1 hypersensitive) immune response (i.e., an allergic
response or reaction) in a subject. Allergens include, but are not
limited to, plant allergens (e.g., pollen, ragweed allergen),
insect allergens, insect sting allergens (e.g., bee sting
allergens), animal allergens (e.g., pet allergens, such as animal
dander or cat Fel d 1 antigen), latex allergens, mold allergens,
fungal allergens, cosmetic allergens, drug allergens, food
allergens, dust, insect venom, viruses, bacteria, etc. Food
allergens include, but are not limited to milk allergens, egg
allergens, nut allergens (e.g., peanut or tree nut allergens, etc.
(e.g., walnuts, cashews, etc.)), fish allergens, shellfish
allergens, soy allergens, legume allergens, seed allergens and
wheat allergens. Insect sting allergens include allergens that are
or are associated with bee stings, wasp stings, hornet stings,
yellow jacket stings, etc. Insect allergens also include house dust
mite allergens (e.g., Der P1 antigen) and cockroach allergens. Drug
allergens include allergens that are or are associated with
antibiotics, NSAIDs, anaesthetics, etc. Pollen allergens include
grass allergens, tree allergens, weed allergens, flower allergens,
etc. Subjects that develop or are at risk of developing an
undesired immune response to any of the allergens provided herein
may be treated with any of the compositions and methods provided
herein. Subjects that may be treated with any of the compositions
and methods provided also include those who have or are at risk of
having an allergy to any of the allergens provided.
[0068] An "allergy" also referred to herein as an "allergic
condition," is any condition where there is an undesired (e.g., a
Type 1 hypersensitive) immune response (i.e., allergic response or
reaction) to a substance. Such substances are referred to herein as
allergens. Allergies or allergic conditions include, but are not
limited to, allergic asthma, hay fever, hives, eczema, plant
allergies, bee sting allergies, pet allergies, latex allergies,
mold allergies, cosmetic allergies, food allergies, allergic
rhinitis or coryza, topic allergic reactions, anaphylaxis, atopic
dermatitis, hypersensitivity reactions and other allergic
conditions. The allergic reaction may be the result of an immune
reaction to any allergen. In some embodiments, the allergy is a
food allergy. Food allergies include, but are not limited to, milk
allergies, egg allergies, nut allergies, fish allergies, shellfish
allergies, soy allergies or wheat allergies.
[0069] "Amount effective" in the context of a composition or dosage
form for administration to a subject refers to an amount of the
composition or dosage form that produces one or more desired immune
responses in the subject, for example, such the stimulation of iNKT
cells to produce IL-4 and/or IL-10. Therefore, in some embodiments,
an amount effective is any amount of a composition provided herein
that produces one or more of these desired immune responses. This
amount can be for in vitro or in vivo purposes. For in vivo
purposes, the amount can be one that a clinician would believe may
have a clinical benefit for a subject in need of tolerization. Such
subjects include those that have or are at risk of having an
inflammatory disease, an autoimmune disease, an allergy, fatty
liver disease, spontaneous abortion, organ or tissue rejection or
graft versus host disease. Such subjects also include those that
have undergone or will undergo transplantation.
[0070] Amounts effective can involve only reducing the level of an
undesired immune response, although in some embodiments, it
involves preventing an undesired immune response altogether.
Amounts effective can also involve delaying the occurrence of an
undesired immune response. An amount that is effective can also be
an amount of a composition provided herein that produces a desired
therapeutic endpoint or a desired therapeutic result. Amounts
effective, preferably, result in tolerance in a subject to an
antigen but in some embodiments the amounts effective simply result
in a tolerogenic effect or a shift to a tolerogenic phenotype. The
achievement of any of the foregoing can be monitored by routine
methods. Preferably, in some embodimenst, the amounts effective
herein are those that result in the production of IL-4 and/or IL-10
by iNKT cells.
[0071] In some embodiments of any of the compositions and methods
provided, the amount effective is one in which the desired immune
response persists in the subject for at least 1 week, at least 2
weeks, at least 1 month, at least 2 months, at least 3 months, at
least 4 months, at least 5 months, at least 6 months, at least 9
months, at least 1 year, at least 2 years, at least 5 years, or
longer. In other embodiments of any of the compositions and methods
provided, the amount effective is one which produces a measurable
desired immune response, for example, a measurable increase or
decrease in an immune response (e.g., to a specific antigen), for
at least 1 week, at least 2 weeks, at least 1 month, at least 2
months, at least 3 months, at least 4 months, at least 5 months, at
least 6 months, at least 9 months, at least 1 year, at least 2
years, at least 5 years, or longer.
[0072] Amounts effective will depend, of course, on the particular
subject being treated; the severity of a condition, disease or
disorder; the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reason.
[0073] In general, doses of the immunosuppressants and/or antigens
in the compositions of the invention can range from about 10
.mu.g/kg to about 100,000 .mu.g/kg. In some embodiments, the doses
can range from about 0.1 mg/kg to about 100 mg/kg. In still other
embodiments, the doses can range from about 0.1 mg/kg to about 25
mg/kg, about 25 mg/kg to about 50 mg/kg, about 50 mg/kg to about 75
mg/kg or about 75 mg/kg to about 100 mg/kg. Alternatively, the dose
can be administered based on the number of synthetic nanocarriers
that provide the desired amount of immunosuppressants and/or
antigens. For example, useful doses include greater than 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9 or 10.sup.10 synthetic nanocarriers
per dose. Other examples of useful doses include from about
1.times.10.sup.6 to about 1.times.10.sup.10, about 1.times.10.sup.7
to about 1.times.10.sup.9 or about 1.times.10.sup.8 to about
1.times.10.sup.9 synthetic nanocarriers per dose.
[0074] "Antigen" means a CD1d-restricted antigen, B cell antigen or
T cell antigen. "Type(s) of antigens" means molecules that share
the same, or substantially the same, antigenic characteristics. In
some embodiments, antigens may be lipids, proteins, polypeptides,
peptides, lipoproteins, glycolipids, polynucleotides,
polysaccharides or are contained or expressed in cells. In some
embodiments, such as when the antigens are not well defined or
characterized, the antigens may be contained within a cell or
tissue preparation, cell debris, cell exosomes, conditioned media,
etc. An antigen can be combined with the synthetic nanocarriers in
the same form as what a subject is exposed to that causes an
undesired immune response but may also be a fragment or derivative
thereof. When a fragment or derivative, however, a desired immune
response to the form encountered by such a subject is the
preferable result with the compositions and methods provided.
[0075] "Antigen-specific" refers to any immune response that
results from the presence of the antigen, or portion thereof, or
that generates molecules that specifically recognize or bind the
antigen.
[0076] "Antigens associated" with a disease, disorder or condition
provided herein are antigens that can generate an undesired immune
response against, as a result of, or in conjunction with the
disease, disorder or condition; the cause of the disease, disorder
or condition (or a symptom or effect thereof); and/or can generate
an undesired immune response that is a symptom, result or effect of
the disease, disorder or condition. Preferably, in some
embodiments, the use of an antigen associated with a disease,
disorder or condition, etc. in the compositions and methods
provided herein will lead to a tolerogenic immune response against
the antigen and/or the cells, by, on or in which the antigen is
expressed. The antigens can be in the same form as expressed in a
subject with the disease, disorder or condition but may also be a
fragment or derivative thereof. When a fragment or derivative,
however, a desired immune response to the form expressed in such a
subject is the preferable result with the compositions and methods
provided.
[0077] In one embodiment, the antigen is an antigen associated with
an inflammatory disease, autoimmune disease, organ or tissue
rejection or graft versus host disease. Such antigens include
autoantigens, such as myelin basic protein, collagen (e.g.,
collagen type 11), human cartilage gp 39, chromogranin A,
gp130-RAPS, proteolipid protein, fibrillarin, nuclear proteins,
nucleolar proteins (e.g., small nucleolar protein), thyroid
stimulating factor receptor, histones, glycoprotein gp 70,
ribosomal proteins, pyruvate dehydrogenase dehydrolipoamide
acetyltransferase, hair follicle antigens, human tropomyosin
isoform 5, mitochondrial proteins, pancreatic .beta.-cell proteins,
myelin oligodendrocyte glycoprotein, insulin, glutamic acid
decarboxylase (GAD), gluten, and fragments or derivatives thereof.
Other autoantigens are provided in Table 1 below.
[0078] Antigens also include those associated with organ or tissue
rejection. Examples of such antigens include, but are not limited
to, antigens from allogeneic cells, e.g., antigens from an
allogeneic cell extract and antigens from other cells, such as
endothelial cell antigens.
[0079] Antigens also include those associated with an allergy. Such
antigens include the allergens described elsewhere herein.
[0080] Antigens also include those associated with a transplantable
graft. Such antigens are associated with (specific for) a
transplantable graft, or an undesired immune response in a
recipient of a transplantable graft that is generated as a result
of the introduction of the transplantable graft in the recipient,
that can be presented for recognition by cells of the immune system
and that can generate an undesired immune response. Transplant
antigens include those associated with organ or tissue rejection or
graft versus host disease. Transplant antigens may be obtained or
derived from cells of a biological material or from information
related to a transplantable graft. Transplant antigens generally
include lipids, proteins, polypeptides, peptides, lipoproteins,
glycolipids, lipids, polynucleotides or are contained or expressed
in cells. Information related to a transplantable graft is any
information about a transplantable graft that can be used to obtain
or derive transplant antigens. Such information includes
information about antigens that would be expected to be present in
or on cells of a transplantable graft such as, for example,
sequence information, types or classes of antigens and/or their
presentation restrictions. Such information may also include
information about the type of transplantable graft (e.g, autograft,
allograft, xenograft), the molecular and cellular composition of
the graft, the bodily location from which the graft is derived or
to which the graft is to be transplanted (e.g., whole or partial
organ, skin, bone, nerves, tendon, neurons, blood vessels, fat,
cornea, etc.).
[0081] "APC presentable antigen" means an antigen that can be
presented for recognition by cells of the immune system, such as
presented by antigen presenting cells, including but not limited to
dendritic cells, B cells or macrophages. The APC presentable
antigen can be presented for recognition by, for example, T cells.
Such antigens may be recognized by and trigger an immune response
in a T cell via presentation of the antigen or portion thereof
bound to a Class I or Class II major histocompatability complex
molecule (MHC), or bound to a CD1d molecule. CD1d is an
antigen-presenting molecule that binds self and foreign lipids and
glycolipids, and is often found on antigen presenting cells. It is
also found on non-hematopoietic cells such as hepatocytes. CD1d
contains a hydrophobic groove which binds hydrophobic lipids,
usually for presentation to iNKT cells. Preferably, one or more
tolerogenic immune responses specific to the APC presentable
antigen results with the compositions provided herein.
[0082] "Assessing an immune response" refers to any measurement or
determination of the level, presence or absence, reduction,
increase in, etc. of an immune response in vitro or in vivo. Such
measurements or determinations may be performed on one or more
samples obtained from a subject. Such assessing can be performed
with any of the methods provided herein or otherwise known in the
art.
[0083] An "at risk" subject is one in which a health practitioner
believes has a chance of having a disease, disorder or condition as
provided herein or is one a health practitioner believes has a
chance of experiencing an undesired immune response as provided
herein.
[0084] An "autoimmune disease" is any disease where the immune
system mounts an undesired immune response against self (e.g., one
or more autoantigens). In some embodiments, an autoimmune disease
comprises an aberrant destruction of cells of the body as part of
the self-targeted immune response. In some embodiments, the
destruction of self manifests in the malfunction of an organ, for
example, the colon or pancreas. Examples of autoimmune diseases are
described elsewhere herein. Additional autoimmune diseases will be
known to those of skill in the art and the invention is not limited
in this respect.
[0085] "Average", as used herein, refers to the arithmetic mean
unless otherwise noted.
[0086] "B cell antigen" means any antigen that triggers an immune
response in a B cell (e.g., an antigen that is specifically
recognized by a B cell or a receptor thereon). In some embodiments,
an antigen that is a T cell antigen is also a B cell antigen. In
other embodiments, the T cell antigen is not also a B cell antigen.
B cell antigens include, but are not limited to proteins, peptides,
small molecules, and carbohydrates. In some embodiments, the B cell
antigen comprises a non-protein antigen (i.e., not a protein or
peptide antigen). In some embodiments, the B cell antigen comprises
a autoantigen. In other embodiments, the B cell antigen is obtained
or derived from an allergen, autoantigen, therapeutic protein, or
transplantable graft.
[0087] "CD1d-restricted antigen" means an antigen that binds to, is
presented by or forms a CD1 complex. CD1d is an MHC-like molecule
with a hydrophobic binding groove which binds lipid (e.g.,
hydrophobic) antigens. The CD1d-restricted antigen can be presented
for recognition by iNKT cells in order to activate them to enhance
tolerogenic immune responses, in some embodiments, in the context
of the compositions of the invention. Generally, CD1d-restricted
antigens are lipids presented to invariant NKT cells. Examples of
CD1d-restricted antigens include lipids and glycolipids and
portions thereof. Specific examples include microbial lipids such
as fatty acids, phospholipids, diacyglycerol, sphingolipids,
glycolipids and lipopeptides. Other examples of CD1d-restricted
antigens include Mycobacterium tuberculosis antigens, such as fatty
acids (e.g., mycolic acids), glycosylated mycolic acids (e.g., GMM,
glucose monomycolate), sulfated acyl trehalose (sulfolipid,
diacylated sulfoglycolipid), lipopeptide dideoxymycobactin (DDM),
isoprenoid based lipids (e.g., MPM, mannosyl-b1-phosphomycoketide),
glycerol based lipids (e.g., PIM's, phosphatidylinositol
mannosides) and hyperglycosylated form of PIMs, mannosylated
lipoarabinomannan (Man-LAM), and mannosyl-b1-phosphoheptaprenol
(MPP); Sphingomonas spp. antigen, such as
.alpha.-glucoronsylceramide (GSL-1); B. burgdorferi antigen, such
as .alpha.-galactosyldiacylglycerol (GalDAG); L. donovani antigen,
such as lipophosphoglycan (LPG); mammalian/self-antigens, such as
phosphatidylinositol (PI), phosphatidylglycerol (PG),
phosphatidylethanolamine (PE), GM1 (a ganglioside), GD3 (a
ganglioside), .beta.-glucosylceramide, sulfatide, and
isoglobotrihexosylceramide (iGB3); synthetic/sponge antigen, such
as .alpha.-galactosylceramide (alpha-GC) or an analog thereof;
.beta.-glucosylceramide, .alpha.-linked glycosphingolipids (from
Sphingomonas spp.), galactosyl diacylglycerols (from Borrelia
burgdorferi), lypophosphoglycan (from Leishmania donovani) and
phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium
leprae), etc. CD1d-restricted antigens can, in some embodiments, be
associated with a disease, disorder or condition as provided
herein. For additional lipids and/or glycolipids useful as a
CD1d-restricted antigens, see V. Cerundolo et al., "Harnessing
invariant NKT cells in vaccination strategies." Nature Rev Immun,
9:28-38 (2009).
[0088] "Concomitantly" means administering two or more substances
to a subject in a manner that is correlated in time, preferably
sufficiently correlated in time so as to provide a modulation in an
immune response. In embodiments, concomitant administration may
occur through administration of two or more substances in the same
dosage form. In other embodiments, concomitant administration may
encompass administration of two or more substances in different
dosage forms, but within a specified period of time, preferably
within 1 month, more preferably within 1 week, still more
preferably within 1 day, and even more preferably within 1
hour.
[0089] "Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling.
[0090] "Derived" means prepared from a material or information
related to a material but is not "obtained" from the material. Such
materials may be substantially modified or processed forms of
materials taken directly from a biological material. Such materials
also include materials produced from information related to a
biological material.
[0091] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject.
[0092] "Encapsulate" means to enclose at least a portion of a
substance within a synthetic nanocarrier. In some embodiments, a
substance is enclosed completely within a synthetic nanocarrier. In
other embodiments, most or all of a substance that is encapsulated
is not exposed to the local environment external to the synthetic
nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%,
10% or 5% (weight/weight) is exposed to the local environment.
Encapsulation is distinct from absorption, which places most or all
of a substance on a surface of a synthetic nanocarrier, and leaves
the substance exposed to the local environment external to the
synthetic nanocarrier.
[0093] "Epitope", also known as an antigenic determinant, is the
part of an antigen that is recognized by the immune system,
specifically by, for example, iNKT cells, antibodies, B cells, or T
cells. In some embodiments, the epitope itself is an antigen.
[0094] A number of epitopes are known to those of skill in the art,
and exemplary epitopes suitable according to some aspects of this
invention include, but are not limited to those listed in the
Immune Epitope Database (www.immuneepitope.org, Vita R, Zarebski L,
Greenbaum J A, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters
B. The immune epitope database 2.0. Nucleic Acids Res. 2010
January; 38(Database issue):D854-62; the entire contents of which
as well as all database entries of IEDB version 2.4, August 2011,
and particularly all epitopes disclosed therein, are incorporated
herein by reference). Epitopes can also be identified with publicly
available algorithms, for example, the algorithms described in Wang
P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010.
peptide binding predictions for HLA DR, DP and DQ molecules. BMC
Bioinformatics 2010, 11:568; Wang P, Sidney J, Dow C, Mothe B,
Sette A, Peters B. 2008. A systematic assessment of MHC class II
peptide binding predictions and evaluation of a consensus approach.
PLoS Comput Biol. 4(4):e1000048; Nielsen M, Lund 0.2009. NN-align.
An artificial neural network-based alignment algorithm for MHC
class II peptide binding prediction. BMC Bioinformatics. 10:296;
Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC class II
binding affinity using SMM-align, a novel stabilization matrix
alignment method. BMC Bioinformatics. 8:238; Bui H H, Sidney J,
Peters B, Sathiamurthy M, Sinichi A, Purton K A, Motile B R,
Chisari F V, Watkins D I, Sette A. 2005. Immunogenetics.
57:304-314; Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O,
Sahin U, Braxenthaler M, Gallazzi F, Protti M P, Sinigaglia F,
Hammer J. 1999. Generation of tissue-specific and promiscuous HLA
ligand databases using DNA microarrays and virtual HLA class II
matrices. Nat. Biotechnol. 17(6):555-561; Nielsen M, Lundegaard C,
Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund O.
2003. Reliable prediction of T-cell epitopes using neural networks
with novel sequence representations. Protein Sci 12:1007-1017; Bui
H H, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton K A,
Mothe B R, Chisari F V, Watkins D I, Sette A. 2005. Automated
generation and evaluation of specific MHC binding predictive tools:
ARB matrix applications. Immunogenetics 57:304-314; Peters B, Sette
A. 2005. Generating quantitative models describing the sequence
specificity of biological processes with the stabilized matrix
method. BMC Bioinformatics 6:132; Chou P Y, Fasman G D. 1978.
Prediction of the secondary structure of proteins from their amino
acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148; Emini E
A, Hughes J V, Perlow D S, Boger J. 1985. Induction of hepatitis A
virus-neutralizing antibody by a virus-specific synthetic peptide.
J Virol 55:836-839; Karplus P A, Schulz G E. 1985. Prediction of
chain flexibility in proteins. Naturwissenschaften 72:212-213;
Kolaskar A S, Tongaonkar P C. 1990. A semi-empirical method for
prediction of antigenic determinants on protein antigens. FEBS
Lett276:172-174; Parker J M, Guo D, Hodges R S. 1986. New
hydrophilicity scale derived from high-performance liquid
chromatography peptide retention data: correlation of predicted
surface residues with antigenicity and X-ray-derived accessible
sites. Biochemistry 25:5425-5432; Larsen J E, Lund O, Nielsen M.
2006. Improved method for predicting linear B-cell epitopes.
Immunome Res 2:2; Ponomarenko J V, Bourne P E. 2007.
Antibody-protein interactions: benchmark datasets and prediction
tools evaluation. BMC Struct Biol 7:64; Haste Andersen P, Nielsen
M, Lund 0.2006. Prediction of residues in discontinuous B-cell
epitopes using protein 3D structures. Protein Sci 15:2558-2567;
Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E, Sette A,
Peters B. 2008. ElliPro: a new structure-based tool for the
prediction of antibody epitopes. BMC Bioinformatics 9:514; Nielsen
M, Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S,
and Lund 0.2008. PLoS Comput Biol.4(7)e1000107. Quantitative
predictions of peptide binding to any HLA-DR molecule of known
sequence: NetMHCIIpan; the entire contents of each of which are
incorporated herein by reference for disclosure of methods and
algorithms for the identification of epitopes.
[0095] "Generating" means causing an action, such as an immune
response (e.g., a tolerogenic immune response) to occur, either
directly oneself or indirectly, such as, but not limited to, an
unrelated third party that takes an action through reliance on
one's words or deeds.
[0096] "Identifying" is any action or set of actions that allows a
clinician to recognize a subject as one who may benefit from the
methods and compositions provided herein. Preferably, the
identified subject is one who is in need of a tolerogenic immune
response as provided herein. The action or set of actions may be
either directly oneself or indirectly, such as, but not limited to,
an unrelated third party that takes an action through reliance on
one's words or deeds.
[0097] "Immunosuppressant" means a compound that causes an APC to
have an immunosuppressive (e.g., tolerogenic effect). An
immunosuppressive effect generally refers to the production or
expression of cytokines or other factors by the APC that reduces,
inhibits or prevents an undesired immune response or that promotes
a desired immune response. When the APC results in an
immunosuppressive effect on immune cells that recognize an antigen
presented by the APC, the immunosuppressive effect is said to be
specific to the presented antigen. Such effect is also referred to
herein as a tolerogenic effect. Without being bound by any
particular theory, it is thought that the immunosuppressive or
tolerogenic effect is a result of the immunosuppressant being
delivered to the APC, preferably in the presence of an antigen,
such as an epitope, (e.g., an administered antigen or one that is
already present in vivo). Accordingly, the immunosuppressant
includes compounds that provide a tolerogenic immune response to an
antigen that may or may not be provided in the same composition or
a different composition. In one embodiment, the immunosuppressant
is one that causes an APC to promote a regulatory phenotype in one
or more immune effector cells. For example, the regulatory
phenotype may be characterized by the production, induction,
stimulation or recruitment of iNKT cells, e.g., such cells that
produce IL-4 and/or IL-10. As another example, the regulatory
phenotype can be characterized in the inhibition of the production,
induction, stimulation or recruitment of iNKT cells that produce
IFN-.gamma.. As another example, the phenotype can be characterized
by iNKT cell stimulation, while in other embodiment, it is
characterized by iNKT cell anergy or the reduction of pathogenic
iNKT cells. The regulatory phenotype, in some embodiments, may be
the induction of FoxP3 in iNKT cells. In one embodiment, the
immunosuppressant is one that affects the response of the APC after
it processes an antigen. In another embodiment, the
immunosuppressant is not one that interferes with the processing of
the antigen. In a further embodiment, the immunosuppressant is not
an apoptotic-signaling molecule. In another embodiment, the
immunosuppressant is not a phospholipid.
[0098] Immunosuppressants include, but are not limited to, statins;
mTOR inhibitors, such as rapamycin or a rapamycin analog;
TGF-.beta. signaling agents; TGF-.beta. receptor agonists; histone
deacetylase inhibitors, such as Trichostatin A; corticosteroids;
inhibitors of mitochondrial function, such as rotenone; P38
inhibitors; NF-.kappa..beta. inhibitors, such as 6Bio,
Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;
prostaglandin E2 agonists (PGE2), such as Misoprostol;
phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor
(PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors;
G-protein coupled receptor agonists; G-protein coupled receptor
antagonists; glucocorticoids; retinoids; cytokine inhibitors;
cytokine receptor inhibitors; cytokine receptor activators;
peroxisome proliferator-activated receptor antagonists; peroxisome
proliferator-activated receptor agonists; histone deacetylase
inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB
inhibitors, such as TGX-221; autophagy inhibitors, such as
3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome
inhibitor I (PSI); and oxidized ATPs, such as P2X receptor
blockers. Immunosuppressants also include IDO, vitamin D3,
cyclosporins, such as cyclosporine A, aryl hydrocarbon receptor
inhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine
(6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol,
mycophenolate mofetil (MMF), aspirin and other COX inhibitors,
niflumic acid, estriol and triptolide. In embodiments, the
immunosuppressant may comprise any of the immunosuppressive agents
provided herein.
[0099] The immunosuppressant can be a compound that directly
provides the immunosuppressive (e.g., tolerogenic) effect on APCs
or it can be a compound that provides the immunosuppressive (e.g.,
tolerogenic) effect indirectly (i.e., after being processed in some
way after administration). Immunosuppressants, therefore, include
prodrug forms of any of the compounds provided herein.
[0100] Immunosuppressants also include nucleic acids that encode
the peptides, polypeptides or proteins provided herein that result
in an immunosuppressive (e.g., tolerogenic) immune response. In
embodiments, therefore, the immunosuppressant is a nucleic acid
that encodes a peptide, polypeptide or protein that results in an
immunosuppressive (e.g., tolerogenic) immune response, and it is
the nucleic acid that is coupled to the synthetic nanocarrier.
[0101] The nucleic acid may be DNA or RNA, such as mRNA. In
embodiments, the inventive compositions comprise a complement, such
as a full-length complement, or a degenerate (due to degeneracy of
the genetic code) of any of the nucleic acids provided herein. In
embodiments, the nucleic acid is an expression vector that can be
transcribed when transfected into a cell line. In embodiments, the
expression vector may comprise a plasmid, retrovirus, or an
adenovirus amongst others. Nucleic acids can be isolated or
synthesized using standard molecular biology approaches, for
example by using a polymerase chain reaction to produce a nucleic
acid fragment, which is then purified and cloned into an expression
vector. Additional techniques useful in the practice of this
invention may be found in Current Protocols in Molecular Biology
2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory
Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer
Institute, Melbourne, Australia; David Russell, University of Texas
Southwestern Medical Center, Dallas, Cold Spring Harbor.
[0102] In embodiments, the immunosuppressants provided herein are
coupled to synthetic nanocarriers. In preferable embodiments, the
immunosuppressant is an element that is in addition to the material
that makes up the structure of the synthetic nanocarrier. For
example, in one embodiment, where the synthetic nanocarrier is made
up of one or more polymers, the immunosuppressant is a compound
that is in addition and coupled to the one or more polymers. As
another example, in one embodiment, where the synthetic nanocarrier
is made up of one or more lipids, the immunosuppressant is again in
addition and coupled to the one or more lipids. In embodiments,
such as where the material of the synthetic nanocarrier also
results in an immunosuppressive (e.g., tolerogenic) effect, the
immunosuppressant is an element present in addition to the material
of the synthetic nanocarrier that results in an immunosuppressive
(e.g., tolerogenic) effect.
[0103] Other exemplary immunosuppressants include, but are not
limited, small molecule drugs, natural products, antibodies (e.g.,
antibodies against CD20, CD3, CD4), biologics-based drugs,
carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense
nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod;
natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506), etc.
Further immunosuppressants, are known to those of skill in the art,
and the invention is not limited in this respect.
[0104] "Inflammatory disease" means any disease, disorder or
condition in which undesired inflammation occurs.
[0105] "Load" of the immunosuppressant or antigen is the amount of
the immunosuppressant or antigen coupled to a synthetic nanocarrier
based on the total weight of materials in an entire synthetic
nanocarrier (weight/weight). Generally, the load is calculated as
an average across a population of synthetic nanocarriers. In one
embodiment, the load of the immunosuppressant on average across the
first population of synthetic nanocarriers is between 0.0001% and
50%. In another embodiment, the load of the antigen on average
across the first and/or second population of synthetic nanocarriers
is between 0.0001% and 50%. In yet another embodiment, the load of
the immunosuppressant and/or antigen is between 0.01% and 20%. In a
further embodiment, the load of the immunosuppressant and/or
antigen is between 0.1% and 10%. In still a further embodiment, the
load of the immunosuppressant and/or antigen is between 1% and 10%.
In yet another embodiment, the load of the immunosuppressant and/or
the antigen is at least 0.1%, at least 0.2%, at least 0.3%, at
least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least
0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at
least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at
least 9%, at least 10%, at least 11%, at least 12%, at least 13%,
at least 14%, at least 15%, at least 16%, at least 17%, at least
18%, at least 19% or at least 20% on average across a population of
synthetic nanocarriers. In yet a further embodiment, the load of
the immunosuppressant and/or the antigen is 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average
across a population of synthetic nanocarriers. In some embodiments
of the above embodiments, the load of the immunosuppressant and/or
the antigen is no more than 25% on average across a population of
synthetic nanocarriers. In embodiments, the load is calculated as
described in the Examples.
[0106] In embodiments of any of the compositions and methods
provided herein, the load may be calculated as follows:
Approximately 3 mg of synthetic nanocarriers are collected and
centrifuged to separate supernatant from synthetic nanocarrier
pellet. Acetonitrile is added to the pellet, and the sample is
sonicated and centrifuged to remove any insoluble material. The
supernatant and pellet are injected on RP-HPLC and absorbance is
read at 278 nm. The .mu.g found in the pellet is used to calculate
% entrapped (load), .mu.g in supernatant and pellet are used to
calculate total .mu.g recovered.
[0107] "Maintenance dose" refers to a dose that is administered to
a subject, after an initial dose has resulted in an
immunosuppressive (e.g., tolerogenic) response in a subject, to
sustain a desired immunosuppressive (e.g., tolerogenic) response. A
maintenance dose, for example, can be one that maintains the
tolerogenic effect achieved after the initial dose, prevents an
undesired immune response in the subject, or prevents the subject
becoming a subject at risk of experiencing an undesired immune
response, including an undesired level of an immune response. In
some embodiments, the maintenance dose is one that is sufficient to
sustain an appropriate level of a desired immune response. In some
embodiments, the maintentance dose is sufficient to sustain an
appropriate level of certain iNKT cell activities or defend against
a challenge with an agent that results in an undesired immune
response.
[0108] "Maximum dimension of a synthetic nanocarrier" means the
largest dimension of a nanocarrier measured along any axis of the
synthetic nanocarrier. "Minimum dimension of a synthetic
nanocarrier" means the smallest dimension of a synthetic
nanocarrier measured along any axis of the synthetic nanocarrier.
For example, for a spheroidal synthetic nanocarrier, the maximum
and minimum dimension of a synthetic nanocarrier would be
substantially identical, and would be the size of its diameter.
Similarly, for a cuboidal synthetic nanocarrier, the minimum
dimension of a synthetic nanocarrier would be the smallest of its
height, width or length, while the maximum dimension of a synthetic
nanocarrier would be the largest of its height, width or length. In
an embodiment, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or greater than 100 nm. In
an embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of inventive synthetic nanocarriers may vary
depending on the embodiment. For instance, aspect ratios of the
maximum to minimum dimensions of the synthetic nanocarriers may
vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1,
more preferably from 1:1 to 10,000:1, more preferably from 1:1 to
1000:1, still more preferably from 1:1 to 100:1, and yet more
preferably from 1:1 to 10:1. Preferably, a maximum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample is equal to or less
than 3 .mu.m, more preferably equal to or less than 2 .mu.m, more
preferably equal to or less than 1 .mu.m, more preferably equal to
or less than 800 nm, more preferably equal to or less than 600 nm,
and more preferably still equal to or less than 500 nm. In
preferred embodiments, a minimum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or greater than
100 nm, more preferably equal to or greater than 120 nm, more
preferably equal to or greater than 130 nm, more preferably equal
to or greater than 140 nm, and more preferably still equal to or
greater than 150 nm. Measurement of synthetic nanocarrier
dimensions (e.g., diameter) is obtained by suspending the synthetic
nanocarriers in a liquid (usually aqueous) media and using dynamic
light scattering (DLS) (e.g. using a Brookhaven ZetaPALS
instrument). For example, a suspension of synthetic nanocarriers
can be diluted from an aqueous buffer into purified water to
achieve a final synthetic nanocarrier suspension concentration of
approximately 0.01 to 0.1 mg/mL. The diluted suspension may be
prepared directly inside, or transferred to, a suitable cuvette for
DLS analysis. The cuvette may then be placed in the DLS, allowed to
equilibrate to the controlled temperature, and then scanned for
sufficient time to acquire a stable and reproducible distribution
based on appropriate inputs for viscosity of the medium and
refractive indicies of the sample. The effective diameter, or mean
of the distribution, is then reported. "Dimension" or "size" or
"diameter" of synthetic nanocarriers means the mean of a particle
size distribution obtained using dynamic light scattering.
[0109] "MHC" refers to major histocompatibility complex, a large
genomic region or gene family found in most vertebrates that
encodes MHC molecules that display fragments or epitopes of
processed proteins on the cell surface. The presentation of
MHC:peptide on cell surfaces allows for surveillance by immune
cells, usually a T cell. There are two general classes of MHC
molecules: Class I and Class II. Generally, Class I MHC molecules
are found on nucleated cells and present peptides to cytotoxic T
cells. Class II MHC molecules are found on certain immune cells,
chiefly macrophages, B cells and dendritic cells, collectively
known as professional APCs. The best-known genes in the MHC region
are the subset that encodes antigen-presenting proteins on the cell
surface. In humans, these genes are referred to as human leukocyte
antigen (HLA) genes.
[0110] "Non-methoxy-terminated polymer" means a polymer that has at
least one terminus that ends with a moiety other than methoxy. In
some embodiments, the polymer has at least two termini that ends
with a moiety other than methoxy. In other embodiments, the polymer
has no termini that ends with methoxy. "Non-methoxy-terminated,
pluronic polymer" means a polymer other than a linear pluronic
polymer with methoxy at both termini. Polymeric nanoparticles as
provided herein can comprise non-methoxy-terminated polymers or
non-methoxy-terminated, pluronic polymers.
[0111] "Obtained" means taken directly from a material and used
with substantially no modification and/or processing.
[0112] "Pharmaceutically acceptable excipient" means a
pharmacologically inactive material used together with the recited
synthetic nanocarriers to formulate the inventive compositions.
Pharmaceutically acceptable excipients comprise a variety of
materials known in the art, including but not limited to
saccharides (such as glucose, lactose, and the like), preservatives
such as antimicrobial agents, reconstitution aids, colorants,
saline (such as phosphate buffered saline), and buffers.
[0113] "Protocol" refers to any dosing regimen of one or more
substances to a subject. A dosing regimen may include the amount,
frequency and/or mode of administration. In some embodiments, such
a protocol may be used to administer one or more compositions of
the invention to one or more test subjects. Immune responses in
these test subject can then be assessed to determine whether or not
the protocol was effective in reducing an undesired immune response
or generating a desired immune response (e.g., the promotion of a
tolerogenic effect). Any other therapeutic and/or prophylactic
effect may also be assessed instead of or in addition to the
aforementioned immune responses. Whether or not a protocol had a
desired effect can be determined using any of the methods provided
herein or otherwise known in the art. For example, a population of
cells may be obtained from a subject to which a composition
provided herein has been administered according to a specific
protocol in order to determine whether or not specific immune
cells, cytokines, antibodies, etc. were reduced, generated,
activated, etc. Useful methods for detecting the presence and/or
number of immune cells include, but are not limited to, flow
cytometric methods (e.g., FACS) and immunohistochemistry methods.
Antibodies and other binding agents for specific staining of immune
cell markers, are commercially available. Such kits typically
include staining reagents for multiple antigens that allow for
FACS-based detection, separation and/or quantitation of a desired
cell population from a heterogeneous population of cells.
[0114] "Providing a subject" is any action or set of actions that
causes a clinician to come in contact with a subject and administer
a composition provided herein thereto or to perform a method
provided herein thereupon. Preferably, the subject is one who is in
need of a tolerogenic immune response as provided herein. The
action or set of actions may be either directly oneself or
indirectly, such as, but not limited to, an unrelated third party
that takes an action through reliance on one's words or deeds.
[0115] "Subject" means animals, including warm blooded mammals such
as humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like.
[0116] "Substantially no B cell epitopes" refers to the absence of
B cell epitopes in an amount (by itself, within the context of the
antigen, in conjunction with a carrier or in conjunction with an
inventive composition) that stimulates substantial activation of a
B cell response. In embodiments, a composition with substantially
no B cell epitopes does not contain a measurable amount of B cell
epitopes of an antigen. In other embodiments, such a composition
may comprise a measurable amount of B cell epitopes of an antigen
but said amount is not effective to generate a measurable B cell
immune response (by itself, within the context of the antigen, in
conjunction with a carrier or in conjunction with an inventive
composition), such as antigen-specific antibody production or
antigen-specific B cell proliferation and/or activity, or is not
effective to generate a significant measurable B cell immune
response (by itself, within the context of the antigen, in
conjunction with a carrier or in conjunction with an inventive
composition). In some embodiments, a significant measurable B cell
immune response is one that produces or would be expected to
produce an adverse clinical result in a subject. In other
embodiments, a significant measurable B cell immune response is one
that is greater than the level of the same type of immune response
(e.g., antigen-specific antibody production or antigen-specific B
cell proliferation and/or activity) produced by a control antigen
(e.g., one known not to comprise B cell epitopes of the antigen or
to stimulate B cell immune responses). In some embodiments, a
significant measurable B cell immune response, such as a
measurement of antibody titers (e.g., by ELISA) is 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold,
20-fold or more greater than the same type of response produced by
a control (e.g., control antigen). In other embodiments, a
composition with substantially no B cell epitopes is one that
produces little to no antigen-specific antibody titers (by itself,
within the context of the antigen, in conjunction with a carrier or
in conjunction with an inventive composition). Such compositions
include those that produce an antibody titer (as an EC50 value) of
less than 500, 400, 300, 200, 100, 50, 40, 30, 20 or 10. In other
embodiments, a significant measurable B cell immune response, is a
measurement of the number or proliferation of B cells that is 10%,
25%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, 15-fold, 20-fold or more greater that the
same type of response produced by a control. Other methods for
measuring B cell responses are known to those of ordinary skill in
the art.
[0117] In embodiments, to ensure that a composition comprises
substantially no B cell epitopes, antigens are selected such that
they do not comprise B cell epitopes for coupling to the synthetic
nanocarriers as provided herein. In other embodiments, to ensure
that a composition comprises substantially no B cell epitopes of an
antigen, the synthetic nanocarriers coupled to the antigen are
produced and tested for B cell immune responses (e.g.,
antigen-specific antibody production, B cell proliferation and/or
activity). Compositions that exhibit the desired properties may
then be selected.
[0118] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, inventive synthetic
nanocarriers do not comprise chitosan. In other embodiments,
inventive synthetic nanocarriers are not lipid-based nanoparticles.
In further embodiments, inventive synthetic nanocarriers do not
comprise a phospholipid.
[0119] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (also referred to
herein as lipid nanoparticles, i.e., nanoparticles where the
majority of the material that makes up their structure are lipids),
polymeric nanoparticles, metallic nanoparticles, surfactant-based
emulsions, dendrimers, buckyballs, nanowires, virus-like particles
(i.e., particles that are primarily made up of viral structural
proteins but that are not infectious or have low infectivity),
peptide or protein-based particles (also referred to herein as
protein particles, i.e., particles where the majority of the
material that makes up their structure are peptides or proteins)
(such as albumin nanoparticles) and/or nanoparticles that are
developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces. Exemplary synthetic nanocarriers
that can be adapted for use in the practice of the present
invention comprise: (1) the biodegradable nanoparticles disclosed
in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric
nanoparticles of Published US Patent Application 20060002852 to
Saltzman et al., (3) the lithographically constructed nanoparticles
of Published US Patent Application 20090028910 to DeSimone et al.,
(4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the
nanoparticles disclosed in Published US Patent Application
2008/0145441 to Penades et al., (6) the protein nanoparticles
disclosed in Published US Patent Application 20090226525 to de los
Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic
acid coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the
nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010), or (11) apoptotic cells, apoptotic bodies or
the synthetic or semisynthetic mimics disclosed in U.S. Publication
2002/0086049. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0120] Synthetic nanocarriers according to the invention that have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
with hydroxyl groups that activate complement or alternatively
comprise a surface that consists essentially of moieties that are
not hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers exclude virus-like
particles. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0121] "T cell antigen" means a CD4+ T-cell antigen, CD8+ cell
antigen or a CD1 d-restricted antigen. "CD4+ T-cell antigen" means
any antigen that is recognized by and triggers an immune response
in a CD4+ T-cell e.g., an antigen that is specifically recognized
by a T-cell receptor on a CD4+ T cell via presentation of the
antigen or portion thereof bound to a Class II major
histocompatability complex molecule (MHC). "CD8+ T cell antigen"
means any antigen that is recognized by and triggers an immune
response in a CD8+ T-cell e.g., an antigen that is specifically
recognized by a T-cell receptor on a CD8+ T cell via presentation
of the antigen or portion thereof bound to a Class I major
histocompatability complex molecule (MHC). "CD1d-restricted
antigen" means an antigen that comprises one or more epitopes, or
portions, that bind to, complex to or are presented by CD1d
molecules. Generally, CD1d-restricted T cell antigens are lipids
presented to invariant NKT cells. CD1d-restricted T cell antigens
may comprise one or more lipids, or glycolipids, including but not
limited to: .alpha.-galactosylceramide (.alpha.-GalCer),
.alpha.-linked glycosphingolipids (from Sphingomonas spp.),
galactosyl diacylglycerols (from Borrelia burgdorferi),
lypophosphoglycan (from Leishmania donovani), endogenous or
exogenous .beta.-glucosylceramide, and phosphatidylinositol
tetramannoside (PIM4) (from Mycobacterium leprae). For additional
lipids and/or glycolipids useful as a CD1d-restricted antigens, see
V. Cerundolo et al., "Harnessing invariant NKT cells in vaccination
strategies." Nature Rev Immun, 9:28-38 (2009).
[0122] "Tolerogenic immune response" means any immune response that
can lead to immune suppression or tolerization, such as against a
CD1d-restricted antigen or an APC presentable antigen (i.e.,
antigen presented by professional or non-professional
antigen-presenting cells) or a cell, tissue, organ, etc. that
expresses such an antigen. Such immune responses include any
reduction, delay or inhibition in an undesired immune response
specific to the antigen or cell, tissue, organ, etc. that expresses
such antigen. Such immune responses also include any stimulation,
production, induction, promotion or recruitment in a desired immune
response specific to the antigen or cell, tissue, organ, etc. that
expresses such antigen. Tolerogenic immune responses, therefore,
include the absence of or reduction in an undesired immune response
to an antigen that can be mediated by antigen reactive cells as
well as the presence or promotion of suppressive cells. Tolerogenic
immune responses as provided herein include immunological
tolerance. To "generate a tolerogenic immune response" refers to
the generation of any of the foregoing immune responses specific to
an antigen or cell, tissue, organ, etc. that expresses such
antigen. The tolerogenic immune response can be the result of MHC
Class I-restricted presentation and/or MHC Class II-restricted
presentation and/or B cell presentation and/or presentation by
CD1d, etc.
[0123] Tolerogenic immune responses include any response that leads
to the stimulation, induction, production or recruitment of iNKT
cells, such as those that produce IL-4 and/or IL-10. Tolerogenic
immune responses also include any response that inhibits the
stimulation, induction, production or recruitment of iNKT cells
that produce IFN-.gamma.. Tolerogenic immune responses also include
any response that leads to the stimulation, induction, production
or recruitment of CD4+ Treg cells cells (e.g., such as CD4+
CD25highFoxP3+ Treg cells) and/or CD8+ Treg cells, which may be
downstream effects of the compositions provided herein (e.g.,
downstream effects as a result of regulatory cytokine production).
In some embodiments, the tolerogenic immune response, is one that
results in the conversion to a regulatory phenotype characterized
by the production, induction, stimulation or recruitment of
regulatory cells or the production of regulatory cytokines.
[0124] CD4+ Treg cells can express the transcription factor FoxP3
and inhibit inflammatory responses and auto-immune inflammatory
diseases (Human regulatory T cells in autoimmune diseases.
Cvetanovich G L, Hafler D A. Curr Opin Immunol. 2010 December;
22(6):753-60. Regulatory T cells and autoimmunity. Vila J, Isaacs J
D, Anderson A E. Curr Opin Hematol. 2009 July; 16(4):274-9). Such
cells also suppress T-cell help to B-cells and induce tolerance to
both self and foreign antigens (Therapeutic approaches to allergy
and autoimmunity based on FoxP3+ regulatory T-cell activation and
expansion. Miyara M, Wing K, Sakaguchi S. J Allergy Clin Immunol.
2009 April; 123(4):749-55). Additionally, FoxP3 may be induced in
other immune cells such as CD8+ T cells, macrophages and iNKT
cells. CD4+ Treg cells recognize antigen when presented by Class II
proteins on APCs. CD8+ Treg cells, which recognize antigen
presented by Class Ib molecule Qa-1, can also suppress T-cell help
to B-cells and result in activation of antigen-specific suppression
inducing tolerance to both self and foreign antigens. Disruption of
the interaction of Qa-1 with CD8+ Treg cells has been shown to
dysregulate immune responses and results in the development of
auto-antibody formation and an auto-immune lethal
systemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16, 467
(7313): 328-32). CD8+ Treg cells have also been shown to inhibit
models of autoimmune inflammatory diseases including rheumatoid
arthritis and colitis (CD4+ CD25+ regulatory T cells in autoimmune
arthritis. Oh S, Rankin A L, Caton A J. Immunol. Rev. 2010 January;
233(1):97-111. Regulatory T cells in inflammatory bowel disease.
Boden E K, Snapper S B. Curr Opin Gastroenterol. 2008 November;
24(6):733-41). In some embodiments, the compositions provided can
effectively result in both types of responses (CD4+ Treg and CD8+
Treg).
[0125] Tolerogenic immune responses also include, but are not
limited to, the induction of regulatory cytokines, such as Treg
cytokines; induction of inhibitory cytokines; the inhibition of
inflammatory cytokines (e.g., IL-4, IL-1b, IL-5, TNF-.alpha., IL-6,
GM-CSF, IFN-.gamma., IL-2, IL-9, IL-12, IL-17, IL-18, IL-21, IL-22,
IL-23, M-CSF, C reactive protein, acute phase protein, chemokines
(e.g., MCP-1, RANTES, MIP-1.alpha., MIP-1.beta., MIG, ITAC or
IP-10), the production of anti-inflammatory cytokines (e.g., IL-4,
IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases
(e.g., MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2),
prostaglandins (e.g., PGE2) or histamines; the inhibition of
polarization to a Th17, Th1 or Th2 immune response; the inhibition
of effector cell-specific cytokines: Th17 (e.g., IL-17, IL-25), Th1
(IFN-.gamma.), Th2 (e.g., IL-4, IL-13); the inhibition of Th1-,
Th2- or Th17-specific transcription factors; the inhibition of
proliferation of effector T cells; the induction of apoptosis of
effector T cells; the induction of tolerogenic dendritic
cell-specific genes; the induction of FoxP3 expression; the
inhibition of IgE induction or IgE-mediated immune responses; the
inhibition of antibody responses (e.g., antigen-specific antibody
production); the inhibition of T helper cell response; the
production of TGF-.beta. and/or IL-10; the inhibition of effector
function of autoantibodies (e.g., inhibition in the depletion of
cells, cell or tissue damage or complement activation); etc. In
some embodiments, the tolerogenic immune response is the
stimulation of iNKT cells. Assessing iNKT cell stimulation includes
analyzing the phenotype, activation and cytokine production of iNKT
cells.
[0126] Any of the foregoing may be measured in vivo in one or more
animal models or may be measured in vitro. One of ordinary skill in
the art is familiar with such in vivo or in vitro measurements.
Undesired immune responses or tolerogenic immune responses can be
monitored using, for example, methods of assessing immune cell
number and/or function, tetramer analysis, ELISPOT, flow
cytometry-based analysis of cytokine expression, cytokine
secretion, cytokine expression profiling, gene expression
profiling, protein expression profiling, analysis of cell surface
markers, PCR-based detection of immune cell receptor gene usage
(see T. Clay et al., "Assays for Monitoring Cellular Immune
Response to Active Immunotherapy of Cancer" Clinical Cancer
Research 7:1127-1135 (2001)), etc. Undesired immune responses or
tolerogenic immune responses may also be monitored using, for
example, methods of assessing protein levels in plasma or serum,
immune cell proliferation and/or functional assays, etc. In some
embodiments, tolerogenic immune responses can be monitored by
assessing the induction of FoxP3. In addition, specific methods are
described in more detail in the Examples.
[0127] Preferably, tolerogenic immune responses lead to the
inhibition of the development, progression or pathology of the
diseases, disorders or conditions described herein. Whether or not
the inventive compositions can lead to the inhibition of the
development, progression or pathology of the diseases, disorders or
conditions described herein can be measured with animal models of
such diseases, disorders or conditions. In some embodiments, the
reduction of an undesired immune response or generation of a
tolerogenic immune response may be assessed by determining clinical
endpoints, clinical efficacy, clinical symptoms, disease biomarkers
and/or clinical scores. Undesired immune responses or tolerogenic
immune responses can also be assessed with diagnostic tests to
assess the presence or absence of a disease, disorder or condition
as provided herein. Undesired immune responses can further be
assessed by methods of measuring proteins levels and/or function in
a subject. In embodiments, methods for monitoring or assessing
undesired allergic responses include assessing an allergic response
in a subject by skin reactivity and/or allergen-specific antibody
production.
[0128] In some embodiments, monitoring or assessing the generation
of an undesired immune response or a tolerogenic immune response in
a subject can be prior to the administration of a composition of
synthetic nanocarriers provided herein and/or prior to
administration of a transplantable graft or exposure to an
allergen. In other embodiments, assessing the generation of an
undesired immune response or tolerogenic immune response can be
after administration of a composition of synthetic nanocarriers
provided herein and/or and after administration of a transplantable
graft or exposure to an allergen. In some embodiments, the
assessment is done after administration of the composition of
synthetic nanocarriers, but prior to administration of a
transplantable graft or exposure to an allergen. In other
embodiments, the assessment is done after administration of a
transplantable graft or exposure to an allergen, but prior to
administration of the composition. In still other embodiments, the
assessment is performed prior to both the administration of the
synthetic nanocarriers and administration of a transplantable graft
or exposure to an allergen, while in yet other embodiments the
assessment is performed after both the administration of synthetic
nanocarriers and the administration of a transplantable graft or
exposure to an allergen. In further embodiments, the assessment is
performed both prior to and after the administration of the
synthetic nanocarriers and/or administration of a transplantable
graft or exposure to an allergen. In still other embodiments, the
assessment is performed more than once on the subject to determine
that a desirable immune state is maintained in the subject, such as
a subject that has or is at risk of having an inflammatory disease,
an autoimmune disease, an allergy or graft verus host disease.
Other subjects include those that have undergone or will undergo
transplantation.
[0129] An antibody response can be assessed by determining one or
more antibody titers. "Antibody titer" means a measurable level of
antibody production. Methods for measuring antibody titers are
known in the art and include Enzyme-linked Immunosorbent Assay
(ELISA). In embodiments, the antibody response can be quantitated,
for example, as the number of antibodies, concentration of
antibodies or titer. The values can be absolute or they can be
relative. Assays for quantifying an antibody response include
antibody capture assays, enzyme-linked immunosorbent assays
(ELISAs), inhibition liquid phase absorption assays (ILPAAs),
rocket immunoelectrophoresis (RIE) assays and line
immunoelectrophoresis (LIE) assays. When an antibody response is
compared to another antibody response the same type of quantitative
value (e.g., titer) and method of measurement (e.g., ELISA) is
preferably used to make the comparison.
[0130] An ELISA method for measuring an antibody titer, for
example, a typical sandwich ELISA, may consist of the following
steps (i) preparing an ELISA-plate coating material such that the
antibody target of interest is coupled to a substrate polymer or
other suitable material (ii) preparing the coating material in an
aqueous solution (such as PBS) and delivering the coating material
solution to the wells of a multiwell plate for overnight deposition
of the coating onto the multiwell plate (iii) thoroughly washing
the multiwell plate with wash buffer (such as 0.05% Tween-20 in
PBS) to remove excess coating material (iv) blocking the plate for
nonspecific binding by applying a diluent solution (such as 10%
fetal bovine serum in PBS), (v) washing the blocking/diluent
solution from the plate with wash buffer (vi) diluting the serum
sample(s) containing antibodies and appropriate standards (positive
controls) with diluent as required to obtain a concentration that
suitably saturates the ELISA response (vii) serially diluting the
plasma samples on the multiwell plate such to cover a range of
concentrations suitable for generating an ELISA response curve
(viii) incubating the plate to provide for antibody-target binding
(ix) washing the plate with wash buffer to remove antibodies not
bound to antigen (x) adding an appropriate concentration of a
secondary detection antibody in same diluent such as a
biotin-coupled detection antibody capable of binding the primary
antibody (xi) incubating the plate with the applied detection
antibody, followed by washing with wash buffer (xii) adding an
enzyme such as streptavidin-HRP (horse radish peroxidase) that will
bind to biotin found on biotinylated antibodies and incubating
(xiii) washing the multiwell plate (xiv) adding substrate(s) (such
as TMB solution) to the plate (xv) applying a stop solution (such
as 2N sulfuric acid) when color development is complete (xvi)
reading optical density of the plate wells at a specific wavelength
for the substrate (450 nm with subtraction of readings at 570 nm)
(xvi) applying a suitable multiparameter curve fit to the data and
defining half-maximal effective concentration (EC50) as the
concentration on the curve at which half the maximum OD value for
the plate standards is achieved.
[0131] A "transplantable graft" refers to a biological material,
such as cells, tissues and organs (in whole or in part) that can be
administered to a subject. Transplantable grafts may be autografts,
allografts, or xenografts of, for example, a biological material
such as an organ, tissue, skin, bone, nerves, tendon, neurons,
blood vessels, fat, cornea, pluripotent cells, differentiated cells
(obtained or derived in vivo or in vitro), etc. In some
embodiments, a transplantable graft is formed, for example, from
cartilage, bone, extracellular matrix, or collagen matrices.
Transplantable grafts may also be single cells, suspensions of
cells and cells in tissues and organs that can be transplanted.
Transplantable cells typically have a therapeutic function, for
example, a function that is lacking or diminished in a recipient
subject. Some non-limiting examples of transplantable cells are
.beta.cells, hepatocytes, hematopoietic stem cells, neuronal stem
cells, neurons, glial cells, or myelinating cells. Transplantable
cells can be cells that are unmodified, for example, cells obtained
from a donor subject and usable in transplantation without any
genetic or epigenetic modifications. In other embodiments,
transplantable cells can be modified cells, for example, cells
obtained from a subject having a genetic defect, in which the
genetic defect has been corrected, or cells that are derived from
reprogrammed cells, for example, differentiated cells derived from
cells obtained from a subject.
[0132] "Transplantation" refers to the process of transferring
(moving) a transplantable graft into a recipient subject (e.g.,
from a donor subject, from an in vitro source (e.g., differentiated
autologous or heterologous native or induced pluripotent cells))
and/or from one bodily location to another bodily location in the
same subject.
[0133] "Undesired immune response" refers to any undesired immune
response that results from exposure to an antigen, promotes or
exacerbates a disease, disorder or condition provided herein (or a
symptom thereof), or is symptomatic of a disease, disorder or
condition provided herein. Such immune responses generally have a
negative impact on a subject's health or is symptomatic of a
negative impact on a subject's health.
C. INVENTIVE COMPOSITIONS
[0134] Provided herein are tolerogenic synthetic nanocarrier
compositions comprising immunosuppressants and CD1d-restricted
antigens and related methods. Such compositions and methods are
useful for reducing the generation of undesired immune responses
and promoting the generation of tolerogenic immune responses by,
for example, stimulating iNKT cells. The compositions provided may
also include other antigens, such as APC presentable antigens. The
compositions provided may also include transplantable grafts. The
compositions may be administered to subjects in which a tolerogenic
immune response is desired. Such subjects include those that have
or are at risk of having an inflammatory disease, an autoimmune
disease, sickle cell disease, fatty liver disease, spontaneous
abortion, an allergy, organ or tissue rejection or graft versus
host disease. Such subjects also include those that have undergone
or will undergo transplantation.
[0135] A wide variety of synthetic nanocarriers can be used
according to the invention. In some embodiments, synthetic
nanocarriers are spheres or spheroids. In some embodiments,
synthetic nanocarriers are flat or plate-shaped. In some
embodiments, synthetic nanocarriers are cubes or cubic. In some
embodiments, synthetic nanocarriers are ovals or ellipses. In some
embodiments, synthetic nanocarriers are cylinders, cones, or
pyramids.
[0136] In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size,
shape, and/or composition so that each synthetic nanocarrier has
similar properties. For example, at least 80%, at least 90%, or at
least 95% of the synthetic nanocarriers, based on the total number
of synthetic nanocarriers, may have a minimum dimension or maximum
dimension that falls within 5%, 10%, or 20% of the average diameter
or average dimension of the synthetic nanocarriers. In some
embodiments, a population of synthetic nanocarriers may be
heterogeneous with respect to size, shape, and/or composition.
[0137] Synthetic nanocarriers can be solid or hollow and can
comprise one or more layers. In some embodiments, each layer has a
unique composition and unique properties relative to the other
layer(s). To give but one example, synthetic nanocarriers may have
a core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
[0138] In some embodiments, synthetic nanocarriers may optionally
comprise one or more lipids. In some embodiments, a synthetic
nanocarrier may comprise a liposome. In some embodiments, a
synthetic nanocarrier may comprise a lipid bilayer. In some
embodiments, a synthetic nanocarrier may comprise a lipid
monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a core comprising a polymeric matrix surrounded by a
lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, a synthetic nanocarrier may comprise a non-polymeric
core (e.g., metal particle, quantum dot, ceramic particle, bone
particle, viral particle, proteins, nucleic acids, carbohydrates,
etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid
monolayer, etc.).
[0139] In other embodiments, synthetic nanocarriers may comprise
metal particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
[0140] In some embodiments, synthetic nanocarriers may optionally
comprise one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
[0141] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In embodiments, the
inventive synthetic nanocarriers do not comprise (or specifically
exclude) carbohydrates, such as a polysaccharide. In certain
embodiments, the carbohydrate may comprise a carbohydrate
derivative such as a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, maltitol, and
lactitol.
[0142] In some embodiments, synthetic nanocarriers can comprise one
or more polymers. In some embodiments, the synthetic nanocarriers
comprise one or more polymers that is a nonmethoxy-terminated,
pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, all
of the polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, the
synthetic nanocarriers comprise one or more polymers that is a
non-methoxy-terminated polymer. In some embodiments, at least 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight)
of the polymers that make up the synthetic nanocarriers are
nonmethoxy-terminated polymers. In some embodiments, all of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated polymers. In some embodiments, the synthetic
nanocarriers comprise one or more polymers that do not comprise
pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polymers that make up
the synthetic nanocarriers do not comprise pluronic polymer. In
some embodiments, all of the polymers that make up the synthetic
nanocarriers do not comprise pluronic polymer. In some embodiments,
such a polymer can be surrounded by a coating layer (e.g.,
liposome, lipid monolayer, micelle, etc.). In some embodiments,
various elements of the synthetic nanocarriers can be coupled with
the polymer.
[0143] The immunosuppressants and/or antigens can be coupled to the
synthetic nanocarriers by any of a number of methods. Generally,
the coupling can be a result of bonding between the
immunosuppressants and/or antigens and the synthetic nanocarrier.
This bonding can result in the immunosuppressants and/or antigens
being attached to the surface of the synthetic nanocarrier and/or
contained within (encapsulated) the synthetic nanocarrier. In some
embodiments, however, the immunosuppressants and/or antigens are
encapsulated by the synthetic nanocarrier as a result of the
structure of the synthetic nanocarrier rather than bonding to the
synthetic nanocarrier. In preferable embodiments, the synthetic
nanocarrier comprises a polymer as provided herein, and the
immunosuppressants and/or antigens are coupled to the polymer.
[0144] When coupling occurs as a result of bonding between the
immunosuppressants and/or antigens and synthetic nanocarriers, the
coupling may occur via a coupling moiety. A coupling moiety can be
any moiety through which an immunosuppressant and/or antigen is
bonded to a synthetic nanocarrier. Such moieties include covalent
bonds, such as an amide bond or ester bond, as well as separate
molecules that bond (covalently or non-covalently) the
immunosuppressant and/or antigen to the synthetic nanocarrier. Such
molecules include linkers or polymers or a unit thereof. For
example, the coupling moiety can comprise a charged polymer to
which an immunosuppressant and/or antigen electrostatically binds.
As another example, the coupling moiety can comprise a polymer or
unit thereof to which it is covalently bonded.
[0145] In preferred embodiments, the synthetic nanocarriers
comprise a polymer as provided herein. These synthetic nanocarriers
can be completely polymeric or they can be a mix of polymers and
other materials.
[0146] In some embodiments, the polymers of a synthetic nanocarrier
associate to form a polymeric matrix. In some of these embodiments,
a component, such as an immunosuppressant or antigen, can be
covalently associated with one or more polymers of the polymeric
matrix. In some embodiments, covalent association is mediated by a
linker. In some embodiments, a component can be noncovalently
associated with one or more polymers of a polymeric matrix. For
example, in some embodiments a component can be encapsulated
within, surrounded by, and/or dispersed throughout a polymeric
matrix. Alternatively or additionally, a component can be
associated with one or more polymers of a polymeric matrix by
hydrophobic interactions, charge interactions, van der Waals
forces, etc. A wide variety of polymers and methods for forming
polymeric matrices therefrom are known conventionally.
[0147] Polymers may be natural or unnatural (synthetic) polymers.
Polymers may be homopolymers or copolymers comprising two or more
monomers. In terms of sequence, copolymers may be random, block, or
comprise a combination of random and block sequences. Typically,
polymers in accordance with the present invention are organic
polymers.
[0148] In some embodiments, the polymer comprises a polyester,
polycarbonate, polyamide, or polyether, or unit thereof. In other
embodiments, the polymer comprises poly(ethylene glycol) (PEG),
polypropylene glycol, poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or a polycaprolactone, or unit
thereof. In some embodiments, it is preferred that the polymer is
biodegradable. Therefore, in these embodiments, it is preferred
that if the polymer comprises a polyether, such as poly(ethylene
glycol) or polypropylene glycol or unit thereof, the polymer
comprises a block-co-polymer of a polyether and a biodegradable
polymer such that the polymer is biodegradable. In other
embodiments, the polymer does not solely comprise a polyether or
unit thereof, such as poly(ethylene glycol) or polypropylene glycol
or unit thereof.
[0149] Other examples of polymers suitable for use in the present
invention include, but are not limited to polyethylenes,
polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.
poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxyacid (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers.
[0150] In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn.177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0151] In some embodiments, polymers can be hydrophilic. For
example, polymers may comprise anionic groups (e.g., phosphate
group, sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g. coupled) within the synthetic nanocarrier.
[0152] In some embodiments, polymers may be modified with one or
more moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
[0153] In some embodiments, polymers may be modified with a lipid
or fatty acid group. In some embodiments, a fatty acid group may be
one or more of butyric, caproic, caprylic, capric, lauric,
myristic, palmitic, stearic, arachidic, behenic, or lignoceric
acid. In some embodiments, a fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0154] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0155] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid:glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0156] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, polycyanoacrylates,
and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
[0157] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene
imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA,
1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo
et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al.,
1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,
Bioconjugate Chem., 4:372) are positively-charged at physiological
pH, form ion pairs with nucleic acids, and mediate transfection in
a variety of cell lines. In embodiments, the inventive synthetic
nanocarriers may not comprise (or may exclude) cationic
polymers.
[0158] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon
et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules,
23:3399). Examples of these polyesters include
poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633).
[0159] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
[0160] In some embodiments, polymers can be linear or branched
polymers. In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that inventive
synthetic nanocarriers may comprise block copolymers, graft
copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
[0161] Compositions according to the invention comprise synthetic
nanocarriers in combination with pharmaceutically acceptable
excipients, such as preservatives, buffers, saline, or phosphate
buffered saline. The compositions may be made using conventional
pharmaceutical manufacturing and compounding techniques to arrive
at useful dosage forms. In an embodiment, inventive synthetic
nanocarriers are suspended in sterile saline solution for injection
together with a preservative.
[0162] In embodiments, when preparing synthetic nanocarriers as
carriers, methods for coupling components to the synthetic
nanocarriers may be useful. If the component is a small molecule it
may be of advantage to attach the component to a polymer prior to
the assembly of the synthetic nanocarriers. In embodiments, it may
also be an advantage to prepare the synthetic nanocarriers with
surface groups that are used to couple the component to the
synthetic nanocarrier through the use of these surface groups
rather than attaching the component to a polymer and then using
this polymer conjugate in the construction of synthetic
nanocarriers.
[0163] In certain embodiments, the coupling can be a covalent
linker. In embodiments, peptides according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with antigen or
immunosuppressant containing an alkyne group or by the 1,3-dipolar
cycloaddition reaction of alkynes on the surface of the nanocarrier
with components containing an azido group. Such cycloaddition
reactions are preferably performed in the presence of a Cu(I)
catalyst along with a suitable Cu(I)-ligand and a reducing agent to
reduce Cu(II) compound to catalytic active Cu(I) compound. This
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0164] Additionally, the covalent coupling may comprise a covalent
linker that comprises an amide linker, a disulfide linker, a
thioether linker, a hydrazone linker, a hydrazide linker, an imine
or oxime linker, an urea or thiourea linker, an amidine linker, an
amine linker, and a sulfonamide linker.
[0165] An amide linker is formed via an amide bond between an amine
on one component with the carboxylic acid group of a second
component such as the nanocarrier. The amide bond in the linker can
be made using any of the conventional amide bond forming reactions
with suitably protected amino acids or components and activated
carboxylic acid such N-hydroxysuccinimide-activated ester.
[0166] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R1--S--S--R2. A disulfide bond can be formed by thiol exchange of a
component containing thiol/mercaptan group(--SH) with another
activated thiol group on a polymer or nanocarrier or a nanocarrier
containing thiol/mercaptan groups with a component containing
activated thiol group.
[0167] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R1 and R2 may be any chemical entities, is made by the
1,3-dipolar cycloaddition reaction of an azide attached to a first
component such as the nanocarrier with a terminal alkyne attached
to a second component such as the immunosuppressant or antigen. The
1,3-dipolar cycloaddition reaction is performed with or without a
catalyst, preferably with Cu(I)-catalyst, which links the two
components through a 1,2,3-triazole function. This chemistry is
described in detail by Sharpless et al., Angew. Chem. Int. Ed.
41(14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108(8),
2952-3015 and is often referred to as a "click" reaction or
CuAAC.
[0168] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The component is
prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The component is then allowed to react with the nanocarrier
via the 1,3-dipolar cycloaddition reaction with or without a
catalyst which covalently couples the component to the particle
through the 1,4-disubstituted 1,2,3-triazole linker.
[0169] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R1-S--R2. Thioether can be made by either alkylation of a
thiol/mercaptan (--SH) group on one component such as the component
with an alkylating group such as halide or epoxide on a second
component such as the nanocarrier. Thioether linkers can also be
formed by Michael addition of a thiol/mercaptan group on one
component to an electron-deficient alkene group on a second
component such as a polymer containing a maleimide group or vinyl
sulfone group as the Michael acceptor. In another way, thioether
linkers can be prepared by the radical thiol-ene reaction of a
thiol/mercaptan group on one component with an alkene group on a
second component such as a polymer or nanocarrier.
[0170] A hydrazone linker is made by the reaction of a hydrazide
group on one component with an aldehyde/ketone group on the second
component such as the nanocarrier.
[0171] A hydrazide linker is formed by the reaction of a hydrazine
group on one component with a carboxylic acid group on the second
component such as the nanocarrier. Such reaction is generally
performed using chemistry similar to the formation of amide bond
where the carboxylic acid is activated with an activating
reagent.
[0172] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component with an
aldehyde or ketone group on the second component such as the
nanocarrier.
[0173] An urea or thiourea linker is prepared by the reaction of an
amine group on one component with an isocyanate or thioisocyanate
group on the second component such as the nanocarrier.
[0174] An amidine linker is prepared by the reaction of an amine
group on one component with an imidoester group on the second
component such as the nanocarrier.
[0175] An amine linker is made by the alkylation reaction of an
amine group on one component with an alkylating group such as
halide, epoxide, or sulfonate ester group on the second component
such as the nanocarrier. Alternatively, an amine linker can also be
made by reductive amination of an amine group on one component with
an aldehyde or ketone group on the second component such as the
nanocarrier with a suitable reducing reagent such as sodium
cyanoborohydride or sodium triacetoxyborohydride.
[0176] A sulfonamide linker is made by the reaction of an amine
group on one component with a sulfonyl halide (such as sulfonyl
chloride) group on the second component such as the
nanocarrier.
[0177] A sulfone linker is made by Michael addition of a
nucleophile to a vinyl sulfone. Either the vinyl sulfone or the
nucleophile may be on the surface of the nanocarrier or attached to
a component.
[0178] The component can also be conjugated to the nanocarrier via
non-covalent conjugation methods. For example, a negative charged
antigen or immunosuppressant can be conjugated to a positive
charged nanocarrier through electrostatic adsorption. A component
containing a metal ligand can also be conjugated to a nanocarrier
containing a metal complex via a metal-ligand complex.
[0179] In embodiments, the component can be attached to a polymer,
for example polylactic acid-block-polyethylene glycol, prior to the
assembly of the synthetic nanocarrier or the synthetic nanocarrier
can be formed with reactive or activatible groups on its surface.
In the latter case, the component may be prepared with a group
which is compatible with the attachment chemistry that is presented
by the synthetic nanocarriers' surface. In other embodiments, a
peptide component can be attached to VLPs or liposomes using a
suitable linker. A linker is a compound or reagent that capable of
coupling two molecules together. In an embodiment, the linker can
be a homobifuntional or heterobifunctional reagent as described in
Hermanson 2008. For example, a VLP or liposome synthetic
nanocarrier containing a carboxylic group on the surface can be
treated with a homobifunctional linker, adipic dihydrazide (ADH),
in the presence of EDC to form the corresponding synthetic
nanocarrier with the ADH linker. The resulting ADH linked synthetic
nanocarrier is then conjugated with a peptide component containing
an acid group via the other end of the ADH linker on NC to produce
the corresponding VLP or liposome peptide conjugate.
[0180] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the component can be coupled by adsorption to a pre-formed
synthetic nanocarrier or it can be coupled by encapsulation during
the formation of the synthetic nanocarrier.
[0181] Any immunosuppressant as provided herein can be coupled to
the synthetic nanocarrier. Immunosuppressants include, but are not
limited to, statins; mTOR inhibitors, such as rapamycin or a
rapamycin analog; TGF-.beta. signaling agents; TGF-.beta. receptor
agonists; histone deacetylase (HDAC) inhibitors; corticosteroids;
inhibitors of mitochondrial function, such as rotenone; P38
inhibitors; NF-.kappa..beta. inhibitors; adenosine receptor
agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors,
such as phosphodiesterase 4 inhibitor; proteasome inhibitors;
kinase inhibitors; G-protein coupled receptor agonists; G-protein
coupled receptor antagonists; glucocorticoids; retinoids; cytokine
inhibitors; cytokine receptor inhibitors; cytokine receptor
activators; peroxisome proliferator-activated receptor antagonists;
peroxisome proliferator-activated receptor agonists; histone
deacetylase inhibitors; calcineurin inhibitors; phosphatase
inhibitors and oxidized ATPs. Immunosuppressants also include IDO,
vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors,
resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic
acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10),
cyclosporine A, siRNAs targeting cytokines or cytokine receptors
and the like.
[0182] Examples of statins include atorvastatin (LIPITOR.RTM.,
TORVAST.RTM.), cerivastatin, fluvastatin (LESCOL.RTM., LESCOL.RTM.
XL), lovastatin (MEVACOR.RTM., ALTOCOR.RTM., ALTOPREV.RTM.),
mevastatin (COMPACTIN.RTM.), pitavastatin (LIVALO.RTM.,
PIAVA.RTM.), rosuvastatin (PRAVACHOL.RTM., SELEKTINE.RTM.,
LIPOSTAT.RTM.), rosuvastatin (CRESTOR.RTM.), and simvastatin
(ZOCOR.RTM., LIPEX.RTM.).
[0183] Examples of mTOR inhibitors include rapamycin and analogs
thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin
(C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap),
C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry
& Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235),
chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus
(RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354
(available from Selleck, Houston, Tex., USA).
[0184] Examples of TGF-.beta. signaling agents include TGF-.beta.
ligands (e.g., activin A, GDF1, GDF11, bone morphogenic proteins,
nodal, TGF-.beta.s) and their receptors (e.g., ACVR1B, ACVR1C,
ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGF.beta.RI, TGF.beta.RII),
R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMADS, SMAD8),
and ligand inhibitors (e.g, follistatin, noggin, chordin, DAN,
lefty, LTBP1, THBS1, Decorin).
[0185] Examples of inhibitors of mitochondrial function include
atractyloside (dipotassium salt), bongkrekic acid (triammonium
salt), carbonyl cyanide m-chlorophenylhydrazone,
carboxyatractyloside (e.g., from Atractylis gummifera), CGP-37157,
(-)-Deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC
binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and
valinomycin (e.g., from Streptomyces fulvissimus) (EMD4Biosciences,
USA).
[0186] Examples of P38 inhibitors include SB-203580
(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)-
, SB-239063
(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-y-
l)imidazole), SB-220025
(5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)-
), and ARRY-797.
[0187] Examples of NF (e.g., NK-.kappa..beta.) inhibitors include
IFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY
11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),
diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132 [
Z-Leu-Leu-Leu-CHO], NF.kappa.B Activation Inhibitor III,
NF-.kappa.B Activation Inhibitor II, JSH-23, parthenolide,
Phenylarsine Oxide (PAO), PPM-18, pyrrolidinedithiocarbamic acid
ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL,
rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132,
sodium salicylate, triptolide (PG490), wedelolactone.
[0188] Examples of adenosine receptor agonists include CGS-21680
and ATL-146e.
[0189] Examples of prostaglandin E2 agonists include E-Prostanoid 2
and E-Prostanoid 4.
[0190] Examples of phosphodiesterase inhibitors (non-selective and
selective inhibitors) include caffeine, aminophylline, IBMX
(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,
theobromine, theophylline, methylated xanthines, vinpocetine, EHNA
(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone
(PERFAN), milrinone, levosimendon, mesembrine, ibudilast,
piclamilast, luteolin, drotaverine, roflumilast (DAXAS.TM.,
DALIRESP.TM.), sildenafil (REVATION.RTM., VIAGRA.RTM.), tadalafil
(ADCIRCA.RTM., CIALIS.RTM.), vardenafil (LEVITRA.RTM.,
STAXYN.RTM.), udenafil, avanafil, icariin, 4-methylpiperazine, and
pyrazolo pyrimidin-7-1.
[0191] Examples of proteasome inhibitors include bortezomib,
disulfuram, epigallocatechin-3-gallate, and salinosporamide A.
[0192] Examples of kinase inhibitors include bevacizumab, BIBW
2992, cetuximab (ERBITUX.RTM.), imatinib (GLEEVEC.RTM.),
trastuzumab (HERCEPTIN.RTM.), gefitinib (IRESSA.RTM.), ranibizumab
(LUCENTIS.RTM.), pegaptanib, sorafenib, dasatinib, sunitinib,
erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080,
pazopanib, mubritinib.
[0193] Examples of glucocorticoids include hydrocortisone
(cortisol), cortisone acetate, prednisone, prednisolone,
methylprednisolone, dexamethasone, betamethasone, triamcinolone,
beclometasone, fludrocortisone acetate, deoxycorticosterone acetate
(DOCA), and aldosterone.
[0194] Examples of retinoids include retinol, retinal, tretinoin
(retinoic acid, RETIN-A.RTM.), isotretinoin (ACCUTANE.RTM.,
AMNESTEEM.RTM., CLARAVIS.RTM., SOTRET.RTM.), alitretinoin
(PANRETIN.RTM.), etretinate (TEGISON) and its metabolite acitretin
(SORIATANE.RTM.), tazarotene (TAZORAC.RTM., AVAGE.RTM.,
ZORAC.RTM.), bexarotene (TARGRETIN.RTM.), and adapalene
(DIFFERIN.RTM.).
[0195] Examples of cytokine inhibitors include IL1ra, IL1 receptor
antagonist, IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin,
Cyclosporin A, Pentamidine, and Pentoxifylline (PENTOPAK.RTM.,
PENTOXIL.RTM., TRENTAL.RTM.).
[0196] Examples of peroxisome proliferator-activated receptor
antagonists include GW9662, PPAR.gamma. antagonist II1, G335,
T0070907 (EMD4Biosciences, USA).
[0197] Examples of peroxisome proliferator-activated receptor
agonists include pioglitazone, ciglitazone, clofibrate, GW1929,
GW7647, L-165,041, LY 171883, PPARy activator, Fmoc-Leu,
troglitazone, and WY-14643 (EMD4Biosciences, USA).
[0198] Examples of histone deacetylase inhibitors include
hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic
tetrapeptides (such as trapoxin B) and depsipeptides, benzamides,
electrophilic ketones, aliphatic acid compounds such as
phenylbutyrate and valproic acid, hydroxamic acids such as
vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat
(LBH589), benzamides such as entinostat (MS-275), CI994, and
mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,
dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.
[0199] Examples of calcineurin inhibitors include cyclosporine,
pimecrolimus, voclosporin, and tacrolimus.
[0200] Examples of phosphatase inhibitors include BN82002
hydrochloride, CP-91149, calyculin A, cantharidic acid,
cantharidin, cypermethrin, ethyl-3,4-dephostatin, fostriecin sodium
salt, MAZ51, methyl-3,4-dephostatin, NSC 95397, norcantharidin,
okadaic acid ammonium salt from prorocentrum concavum, okadaic
acid, okadaic acid potassium salt, okadaic acid sodium salt,
phenylarsine oxide, various phosphatase inhibitor cocktails,
protein phosphatase 1C, protein phosphatase 2A inhibitor protein,
protein phosphatase 2A1, protein phosphatase 2A2, sodium
orthovanadate.
[0201] In some embodiments, antigens as described herein are
coupled to synthetic nanocarriers. In some embodiments, the
antigens are coupled to the same or different synthetic
nanocarriers as to which the immunosuppressants are coupled. In
other embodiments, the antigens are not coupled to any synthetic
nanocarriers. Antigens include those described above including
antigens associated with an inflammatory disease, autoimmune
disease, allergy, fatty liver disease, spontaneous abortion, organ
or tissue rejection or graft versus host disease, a transplantable
graft, etc. The full length antigens themselves can be coupled to
the synthetic nanocarriers. Fragments or derivatives of any of the
foregoing that include epitopes can also be coupled to the
synthetic nanocarriers.
[0202] In some embodiments, a component, such as an antigen or
immunosuppressant, may be isolated. Isolated refers to the element
being separated from its native environment and present in
sufficient quantities to permit its identification or use. This
means, for example, the element may be (i) selectively produced by
expression cloning or (ii) purified as by chromatography or
electrophoresis. Isolated elements may be, but need not be,
substantially pure. Because an isolated element may be admixed with
a pharmaceutically acceptable excipient in a pharmaceutical
preparation, the element may comprise only a small percentage by
weight of the preparation. The element is nonetheless isolated in
that it has been separated from the substances with which it may be
associated in living systems, i.e., isolated from other lipids or
proteins. Any of the elements provided herein may be isolated. Any
of the antigens provided herein can be included in the compositions
in isolated form.
D. METHODS OF MAKING AND USING THE INVENTIVE COMPOSITIONS AND
RELATED METHODS
[0203] Synthetic nanocarriers may be prepared using a wide variety
of methods known in the art. For example, synthetic nanocarriers
can be formed by methods as nanoprecipitation, flow focusing
fluidic channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0204] Various materials may be encapsulated into synthetic
nanocarriers as desirable using a variety of methods including but
not limited to C. Astete et al., "Synthesis and characterization of
PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3,
pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials into synthetic nanocarriers
may be used, including without limitation methods disclosed in U.S.
Pat. No. 6,632,671 to Unger Oct. 14, 2003.
[0205] In certain embodiments, synthetic nanocarriers are prepared
by a nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
[0206] If particles prepared by any of the above methods have a
size range outside of the desired range, particles can be sized,
for example, using a sieve.
[0207] Elements (i.e., components) of the inventive synthetic
nanocarriers (such as moieties of which an immunofeature surface is
comprised, targeting moieties, polymeric matrices, antigens,
immunosuppressants and the like) may be coupled to the overall
synthetic nanocarrier, e.g., by one or more covalent bonds, or may
be coupled by means of one or more linkers. Additional methods of
functionalizing synthetic nanocarriers may be adapted from
Published US Patent Application 2006/0002852 to Saltzman et al.,
Published US Patent Application 2009/0028910 to DeSimone et al., or
Published International Patent Application WO/2008/127532 A1 to
Murthy et al.
[0208] Alternatively or additionally, synthetic nanocarriers can be
coupled to components directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
coupling is mediated by non-covalent interactions including but not
limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such couplings may be
arranged to be on an external surface or an internal surface of an
inventive synthetic nanocarrier. In embodiments, encapsulation
and/or absorption is a form of coupling. In embodiments, the
inventive synthetic nanocarriers can be combined with an antigen by
admixing in the same vehicle or delivery vehicle system.
[0209] Populations of synthetic nanocarriers may be combined to
form pharmaceutical dosage forms according to the present invention
using traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, each
containing one or more subsets of nanocarriers, are directly
combined or are brought together via one or more vessels containing
diluent. As synthetic nanocarriers may also be produced or stored
in a powder form, dry powder-powder mixing could be performed as
could the re-suspension of two or more powders in a common media.
Depending on the properties of the nanocarriers and their
interaction potentials, there may be advantages conferred to one or
another route of mixing.
[0210] Typical inventive compositions that comprise synthetic
nanocarriers may comprise inorganic or organic buffers (e.g.,
sodium or potassium salts of phosphate, carbonate, acetate, or
citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium
or potassium hydroxide, salts of citrate or acetate, amino acids
and their salts) antioxidants (e.g., ascorbic acid,
alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate
80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate),
solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose,
mannitol, trehalose), osmotic adjustment agents (e.g., salts or
sugars), antibacterial agents (e.g., benzoic acid, phenol,
gentamicin), antifoaming agents (e.g., polydimethylsilozone),
preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric
stabilizers and viscosity-adjustment agents (e.g.,
polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and
co-solvents (e.g., glycerol, polyethylene glycol, ethanol).
[0211] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. Techniques suitable
for use in practicing the present invention may be found in
Handbook of Industrial Mixing: Science and Practice, Edited by
Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004
John Wiley & Sons, Inc.; and Pharmaceutics: The Science of
Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill
Livingstone. In an embodiment, inventive synthetic nanocarriers are
suspended in sterile saline solution for injection together with a
preservative.
[0212] It is to be understood that the compositions of the
invention can be made in any suitable manner, and the invention is
in no way limited to compositions that can be produced using the
methods described herein. Selection of an appropriate method may
require attention to the properties of the particular moieties
being associated.
[0213] In some embodiments, inventive synthetic nanocarriers are
manufactured under sterile conditions or are terminally sterilized.
This can ensure that resulting compositions are sterile and
non-infectious, thus improving safety when compared to non-sterile
compositions. This provides a valuable safety measure, especially
when subjects receiving synthetic nanocarriers have immune defects,
are suffering from infection, and/or are susceptible to infection.
In some embodiments, inventive synthetic nanocarriers may be
lyophilized and stored in suspension or as lyophilized powder
depending on the formulation strategy for extended periods without
losing activity.
[0214] The compositions of the invention can be administered by a
variety of routes, including but not limited to subcutaneous,
intranasal, oral, intravenous, intraperitoneal, intramuscular,
transmuco sal, transmucosal, sublingual, rectal, ophthalmic,
pulmonary, intradermal, transdermal, transcutaneous or intradermal
or by a combination of these routes. Routes of administration also
include administration by inhalation or pulmonary aerosol.
Techniques for preparing aerosol delivery systems are well known to
those of skill in the art (see, for example, Sciarra and Cutie,
"Aerosols," in Remington's Pharmaceutical Sciences, 18th edition,
1990, pp. 1694-1712; incorporated by reference).
[0215] The transplantable grafts provided as a cell-based therapy
of the invention may be administered by parenteral, intraarterial,
intranasal or intravenous administration or by injection to lymph
nodes or anterior chamber of the eye or by local administration to
an organ or tissue of interest. The administration may be by
subcutaneous, intrathecal, intraventricular, intramuscular,
intraperitoneal, intracoronary, intrapancreatic, intrahepatic or
bronchial injection.
[0216] The compositions of the invention can be administered in
effective amounts, such as the effective amounts described
elsewhere herein. Doses of dosage forms contain varying amounts of
populations of synthetic nanocarriers and/or varying amounts of
immunosuppressants and/or antigens, according to the invention. The
amount of synthetic nanocarriers and/or immunosuppressants and/or
antigens present in the inventive dosage forms can be varied
according to the nature of the antigens, the therapeutic benefit to
be accomplished, and other such parameters. In embodiments, dose
ranging studies can be conducted to establish optimal therapeutic
amount of the population of synthetic nanocarriers and the amount
of immunosuppressants and/or antigens to be present in the dosage
form. In embodiments, the synthetic nanocarriers and/or the
immunosuppressants and/or antigens are present in the dosage form
in an amount effective to generate a tolerogenic immune response to
the antigens upon administration to a subject. It may be possible
to determine amounts of the immunosuppressants and/or antigens
effective to generate a tolerogenic immune response using
conventional dose ranging studies and techniques in subjects.
Inventive dosage forms may be administered at a variety of
frequencies. In a preferred embodiment, at least one administration
of the dosage form is sufficient to generate a pharmacologically
relevant response. In more preferred embodiment, at least two
administrations, at least three administrations, or at least four
administrations, of the dosage form are utilized to ensure a
pharmacologically relevant response.
[0217] Prophylactic administration of the inventive compositions
can be initiated prior to the onset of disease, disorder or
condition or therapeutic administration can be initiated after a
disorder, disorder or condition is established.
[0218] In some embodiments, administration of synthetic
nanocarriers is undertaken e.g., prior to administration of a
transplantable graft or exposure to an allergen. In exemplary
embodiments, synthetic nanocarriers are administered at one or more
times including, but not limited to, 30, 25, 20, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days prior to
administration of a transplantable graft or exposure to an
allergen. In addition or alternatively, synthetic nanocarriers can
be administered to a subject following administration of a
transplantable graft or exposure to an allergen. In exemplary
embodiments, synthetic nanocarriers are administered at one or more
times including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 20, 25, 30, etc. days following administration
of a transplantable graft or exposure to an allergen.
[0219] In some embodiments, a maintenance dose (e.g., of a
synthetic nanocarrier composition provided herein) is administered
to a subject after an initial administration has resulted in a
tolerogenic response in the subject, for example to maintain the
tolerogenic effect achieved after the initial dose, to prevent an
undesired immune reaction in the subject, or to prevent the subject
becoming a subject at risk of experiencing an undesired immune
response or an undesired level of an immune response. In some
embodiments, the maintenance dose is the same dose as the initial
dose the subject received. In some embodiments, the maintenance
dose is a lower dose than the initial dose. For example, in some
embodiments, the maintenance dose is about 3/4, about 2/3, about
1/2, about 1/3, about 1/4, about 1/8, about 1/10, about 1/20, about
1/25, about 1/50, about 1/100, about 1/1,000, about 1/10,000, about
1/100,000, or about 1/1,000,000 (weight/weight) of the initial
dose.
[0220] The compositions and methods described herein can be used to
induce or enhance a tolerogenic immune response and/or to suppress,
modulate, direct or redirect an undesired immune response for the
purpose of immune suppression. The compositions and methods
described herein can be used in the diagnosis, prophylaxis and/or
treatment of diseases, disorders or conditions in which immune
suppression (e.g., tolerogenic immune response) would confer a
treatment benefit. Such diseases, disorders or conditions include
inflammatory diseases, autoimmune diseases, allergies, sickle cell
disease, fatty liver disease, spontaneous abortion, organ or tissue
rejection and graft versus host disease. The compositions and
methods described herein can also be used in subjects who have
undergone or will undergo transplantation.
[0221] Autoimmune diseases include, but are not limited to,
rheumatoid arthritis, multiple sclerosis, immune-mediated or Type I
diabetes mellitus, inflammatory bowel disease (e.g., Crohn's
disease or ulcerative colitis), systemic lupus erythematosus,
psoriasis, scleroderma, autoimmune thyroid disease, alopecia
greata, Grave's disease, Guillain-Barre syndrome, celiac disease,
Sjogren's syndrome, rheumatic fever, gastritis, autoimmune atrophic
gastritis, autoimmune hepatitis, insulitis, oophoritis, orchitis,
uveitis, phacogenic uveitis, myasthenia gravis, primary myxoedema,
pernicious anemia, autoimmune haemolytic anemia, Addison's disease,
scleroderma, Goodpasture's syndrome, nephritis, for example,
glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,
sympathetic opthalmia, idiopathic thrombocylopenic purpura,
idiopathic feucopenia, Wegener's granulomatosis and
poly/dermatomyositis.
[0222] Some additional exemplary autoimmune diseases, associated
autoantigens, and autoantibodies, which are contemplated for use in
the invention, are described in Table 1 below:
TABLE-US-00001 Autoantibody Type Autoantibody Autoantigen
Autoimmune disease or disorder Antinuclear Anti-SSA/Ro
ribonucleoproteins Systemic lupus erythematosus, neonatal
antibodies autoantibodies heart block, primary Sjogren's syndrome
Anti-La/SS-B ribonucleoproteins Primary Sjogren's syndrome
autoantibodies Anti-centromere centromere CREST syndrome antibodies
Anti-neuronal Ri[disambiguation Opsoclonus nuclear antibody-2
needed] Anti-dsDNA double-stranded SLE DNA Anti-Jo1 histidine-tRNA
Inflammatory myopathy ligase Anti-Smith snRNP core proteins SLE
Anti- Type I Systemic sclerosis (anti-Scl-70 antibodies)
topoisomerase topoisomerase antibodies Anti-histone histones SLE
and Drug-induced LE[2] antibodies Anti-p62 nucleoporin 62 Primary
biliary cirrhosis[3][4][5] antibodies[3] Anti-sp100 Sp100 nuclear
antibodies [4] antigen Anti-glycoprotein- nucleoporin 210 kDa 210
antibodies[5] Anti- Anti-tTG Coeliac disease transglutaminase
Anti-eTG Dermatitis herpetiformis antibodies Anti-ganglioside
ganglioside GQ1B Miller-Fisher Syndrome antibodies ganglioside GD3
Acute motor axonal neuropathy (AMAN) ganglioside GM1 Multifocal
motor neuropathy with conduction block (MMN) Anti-actin actin
Coeliac disease anti-actin antibodies antibodies correlated with
the level of intestinal damage [6][7] Liver kidney Autoimmune
hepatitis.[8] microsomal type 1 antibody Lupus anticoagulant
Anti-thrombin thrombin Systemic lupus erythematosus antibodies
Anti-neutrophil phospholipid Antiphospholipid syndrome cytoplasmic
c-ANCA proteins in Wegener's granulomatosis antibody neutrophil
cytoplasm p-ANCA neutrophil Microscopic polyangiitis, Churg-Strauss
perinuclear syndrome, systemic vasculitides (non- specific)
Rheumatoid factor IgG Rheumatoid arthritis Anti-smooth muscle
smooth muscle Chronic autoimmune hepatitis antibody
Anti-mitochondrial mitochondria Primary biliary cirrhosis[9]
antibody Anti-SRP signal recognition Polymyositis[10] particle
exosome complex Scleromyositis nicotinic Myasthenia gravis
acetylcholine receptor muscle-specific Myasthenia gravis kinase
(MUSK) Anti-VGCC voltage-gated Lambert-Eaton myasthenic syndrome
calcium channel (P/Q-type) thyroid peroxidase Hashimoto's
thyroiditis (microsomal) TSH receptor Graves' disease Hu
Paraneoplastic cerebellar syndrome Yo (cerebellar Paraneoplastic
cerebellar syndrome Purkinje Cells) amphiphysin Stiff person
syndrome, paraneoplastic cerebellar syndrome Anti-VGKC
voltage-gated Limbic encephalitis, Isaac's Syndrome potassium
channel (autoimmune neuromyotonia) (VGKC) basal ganglia Sydenham's
chorea, paediatric autoimmune neurons neuropsychiatric disease
associated with Streptococcus (PANDAS) N-methyl-D- Encephalitis
aspartate receptor (NMDA) glutamic acid Diabetes mellitus type 1,
stiff person decarboxylase syndrome (GAD) aquaporin-4 Neuromyelitis
optica (Devic's syndrome)
[0223] Inflammatory diseases include, but are not limited to,
Alzheimer's, Ankylosing spondylitis, arthritis, asthma,
atherosclerosis, Behcet's disease, chronic inflammatory
demyelinating polyradiculoneuropathy, Crohn's disease, colitis,
cystic fibrosis, dermatitis, diverticulitis, hepatitis, irritable
bowel syndrome (IBS), lupus erythematous, muscular dystrophy,
nephritis, Parkinson's, shingles and ulcerative colitis.
Inflammatory diseases also include, for example, cardiovascular
disease, chronic obstructive pulmonary disease (COPD),
bronchiectasis, chronic cholecystitis, tuberculosis, Hashimoto's
thyroiditis, sepsis, sarcoidosis, silicosis and other
pneumoconioses, and an implanted foreign body in a wound, but are
not so limited. As used herein, the term "sepsis" refers to a
well-recognized clinical syndrome associated with a host's systemic
inflammatory response to microbial invasion. The term "sepsis" as
used herein refers to a condition that is typically signaled by
fever or hypothermia, tachycardia, and tachypnea, and in severe
instances can progress to hypotension, organ dysfunction, and even
death.
[0224] In some embodiments, the inflammatory disease is
non-autoimmune inflammatory bowel disease, post-surgical adhesions,
coronary artery disease, hepatic fibrosis, acute respiratory
distress syndrome, acute inflammatory pancreatitis, endoscopic
retrograde cholangiopancreatography-induced pancreatitis, burns,
atherogenesis of coronary, cerebral and peripheral arteries,
appendicitis, cholecystitis, diverticulitis, visceral fibrotic
disorders, wound healing, skin scarring disorders (keloids,
hidradenitis suppurativa), granulomatous disorders (sarcoidosis,
primary biliary cirrhosis), asthma, pyoderma gandrenosum, Sweet's
syndrome, Behcet's disease, primary sclerosing cholangitis or an
abscess. In some preferred embodiment the inflammatory disease is
inflammatory bowel disease (e.g., Crohn's disease or ulcerative
colitis).
[0225] In other embodiments, the inflammatory disease is an
autoimmune disease. The autoimmune disease in some embodiments is
rheumatoid arthritis, rheumatic fever, ulcerative colitis, Crohn's
disease, autoimmune inflammatory bowel disease, insulin-dependent
diabetes mellitus, diabetes mellitus, juvenile diabetes,
spontaneous autoimmune diabetes, gastritis, autoimmune atrophic
gastritis, autoimmune hepatitis, thyroiditis, Hashimoto's
thyroiditis, insulitis, oophoritis, orchitis, uveitis, phacogenic
uveitis, multiple sclerosis, myasthenia gravis, primary myxoedema,
thyrotoxicosis, pernicious anemia, autoimmune haemolytic anemia,
Addison's disease, Anklosing spondylitis, sarcoidosis, scleroderma,
Goodpasture's syndrome, Guillain-Barre syndrome, Graves' disease,
glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,
excema, bulous pemiphigous, sympathetic opthalmia, idiopathic
thrombocylopenic purpura, idiopathic feucopenia, Sjogren's
syndrome, systemic sclerosis, Wegener's granulomatosis,
poly/dermatomyositis, primary biliary cirrhosis, primary sclerosing
cholangitis, lupus or systemic lupus erythematosus.
[0226] Graft versus host disease (GVHD) is a complication that can
occur after a pluripotent cell (e.g., stem cell) or bone marrow
transplant in which the newly transplanted material results in an
attack on the transplant recipient's body. In some instances, GVHD
takes place after a blood transfusion. Graft-versus-host-disease
can be divided into acute and chronic forms. The acute or fulminant
form of the disease (aGVHD) is normally observed within the first
100 days post-transplant, and is a major challenge to transplants
owing to associated morbidity and mortality. The chronic form of
graft-versus-host-disease (cGVHD) normally occurs after 100 days.
The appearance of moderate to severe cases of cGVHD adversely
influences long-term survival.
EXAMPLES
Example 1
Immune Response of Synthetic Nanocarriers with Coupled Rapamycin
With and without Ovalbumin Peptide (323-339)
Materials
[0227] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson
Street, Framingham, Mass. 01702; Product Catalogue # R1017). PLGA
with a lactide:glycolide ratio of 3:1 and an inherent viscosity of
0.75 dL/g was purchased from SurModics Pharmaceuticals (756 Tom
Martin Drive, Birmingham, Ala. 35211; Product Code 7525 DLG 7A).
Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD
Chemicals (Product Number 1.41350.1001).
[0228] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature.
[0229] Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The
solution was prepared by dissolving rapamycin in pure methylene
chloride.
[0230] Solution 3: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0231] Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0232] Method for Preparing Synthetic Nanocarrier Containing
Rapamycin and Ovalbumin (323-339)
[0233] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.2 mL),
and solution 3 (1.0 mL) in a small pressure tube and sonicating at
50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
A secondary emulsion (W1/O1/W2) was then prepared by combining
solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexing for
10 s, and sonicating at 30% amplitude for 60 seconds using the
Branson Digital Sonifier 250.
[0234] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the synthetic nanocarriers to form. A portion of
the synthetic nanocarriers were washed by transferring the
synthetic nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final synthetic
nanocarrier dispersion of about 10 mg/mL.
[0235] The amounts of peptide and rapamycin in the synthetic
nanocarriers were determined by HPLC analysis. The total
dry-synthetic nanocarrier mass per mL of suspension was determined
by a gravimetric method.
Method for Synthetic Nanocarrier Containing Rapamycin
[0236] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining 0.13 M hydrochloric acid solution (0.2
mL), solution 2 (0.2 mL), and solution 3 (1.0 mL) in a small
pressure tube and sonicating at 50% amplitude for 40 seconds using
a Branson Digital Sonifier 250. A secondary emulsion (W1/O1/W2) was
then prepared by combining solution 4 (3.0 mL) with the primary
W1/O1 emulsion, vortexing for 10 s, and sonicating at 30% amplitude
for 60 seconds using the Branson Digital Sonifier 250.
[0237] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the synthetic nanocarriers to form. A portion of
the synthetic nanocarriers were washed by transferring the
synthetic nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final synthetic
nanocarrier dispersion of about 10 mg/mL.
[0238] The amount of rapamycin in the synthetic nanocarrier was
determined by HPLC analysis. The total dry-synthetic nanocarrier
mass per mL of suspension was determined by a gravimetric
method.
Method for Measuring Rapamycin Load
[0239] Approximately 3 mg of synthetic nanocarriers were collected
and centrifuged to separate supernatant from synthetic nanocarrier
pellet. Acetonitrile was added to the pellet, and the sample was
sonicated and centrifuged to remove any insoluble material. The
supernatant and pellet were injected on RP-HPLC and absorbance was
read at 278 nm. The .mu.g found in the pellet were used to
calculate % entrapped (load), .mu.g in supernatant and pellet were
used to calculate total .mu.g recovered.
Method for Measuring Ovalbumin (323-339) Load
[0240] Approximately 3 mg of synthetic nanocarriers were collected
and centrifuged to separate supernatant from synthetic nanocarrier
pellet. Trifluoroethanol was added to the pellet and the sample was
sonicated to dissolve the polymer, 0.2% trifluoroacetic acid was
added and sample was sonicated and then centrifuged to remove any
insoluble material. The supernatant and pellet were injected on
RP-HPLC and absorbance was read at 215 nm. The .mu.g found in the
pellet were used to calculate % entrapped (load), .mu.g in
supernatant and pellet were used to calculate total .mu.g
recovered.
Antigen-Specific Tolerogenic Dendritic Cells (tDC) Activity on Treg
Cell Development
[0241] The assay included the use of OTII mice which have a
transgenic T-cell receptor specific for an immune-dominant
ovalbumin (323-339). In order to create antigen-specific tDCs,
CD11c+ splenocytes were isolated, and the ovalbumin (323-339)
peptide added in vitro at 1 .mu.g/ml or no antigen. Soluble or
nanocarrier-encapsulated rapamycin was then added to the DCs for 2
hours which were then washed extensively to remove free rapamycin
from the culture. Purified responder CD4+ CD25- cells were isolated
from DT11 mice and added to tDC at a 10:1 T to DC ratio. The
mixture of tDC and OTII T-cells were then cultured for 4-5 days,
and the frequency of Treg cells (CD4+ CD25highFoxP3+) were analyzed
by flow cytometry as shown in FIG. 1. Regions were selected based
on isotype controls.
Example 2
Mesoporous Silica Nanoparticles with Coupled Ibuprofen
(Prophetic)
[0242] Mesoporous SiO2 nanoparticle cores are created through a
sol-gel process. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g)
is dissolved in deionized water (500 mL), and then 2 M aqueous NaOH
solution (3.5 mL) is added to the CTAB solution. The solution is
stirred for 30 min, and then Tetraethoxysilane (TEOS) (2.5 mL) is
added to the solution. The resulting gel is stirred for 3 h at a
temperature of 80.degree. C. The white precipitate which forms is
captured by filtration, followed by washing with deionized water
and drying at room temperature. The remaining surfactant is then
extracted from the particles by suspension in an ethanolic solution
of HCl overnight. The particles are washed with ethanol,
centrifuged, and redispersed under ultrasonication. This wash
procedure is repeated two additional times.
[0243] The SiO2 nanoparticles are then functionalized with amino
groups using (3-aminopropyl)-triethoxysilane (APTMS). To do this,
the particles are suspended in ethanol (30 mL), and APTMS (50
.mu.L) is added to the suspension. The suspension is allowed to
stand at room temperature for 2 h and then is boiled for 4 h,
keeping the volume constant by periodically adding ethanol.
Remaining reactants are removed by five cycles of washing by
centrifugation and redispersing in pure ethanol.
[0244] In a separate reaction, 1-4 nm diameter gold seeds are
created. All water used in this reaction is first deionized and
then distilled from glass. Water (45.5 mL) is added to a 100 mL
round-bottom flask. While stirring, 0.2 M aqueous NaOH (1.5 mL) is
added, followed by a 1% aqueous solution of
tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL). Two
minutes after the addition of THPC solution, a 10 mg/mL aqueous
solution of chloroauric acid (2 mL), which has been aged at least
15 min, is added. The gold seeds are purified through dialysis
against water.
[0245] To form the core-shell nanocarriers, the
amino-functionalized SiO2 nanoparticles formed above are first
mixed with the gold seeds for 2 h at room temperature. The
gold-decorated SiO2 particles are collected through centrifugation
and mixed with an aqueous solution of chloroauric acid and
potassium bicarbonate to form the gold shell. The particles are
then washed by centrifugation and redispersed in water. Ibuprofen
is loaded by suspending the particles in a solution of sodium
ibuprofen (1 mg/L) for 72 h. Free ibuprofen is then washed from the
particles by centrifugation and redispersing in water.
Example 3
Liposomes Containing Cyclosporine A (Prophetic)
[0246] The liposomes are formed using thin film hydration.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 .mu.mol),
cholesterol (32 .mu.mol), and cyclosporin A (6.4 .mu.mol) are
dissolved in pure chloroform (3 mL). This lipid solution is added
to a 50 mL round-bottom flask, and the solvent is evaporated on a
rotary evaporator at a temperature of 60.degree. C. The flask is
then flushed with nitrogen gas to remove remaining solvent.
Phosphate buffered saline (2 mL) and five glass beads are added to
the flask, and the lipid film is hydrated by shaking at 60.degree.
C. for 1 h to form a suspension. The suspension is transferred to a
small pressure tube and sonicated at 60.degree. C. for four cycles
of 30s pulses with a 30 s delay between each pulse. The suspension
is then left undisturbed at room temperature for 2 h to allow for
complete hydration. The liposomes are washed by centrifugation
followed by resuspension in fresh phosphate buffered saline.
Example 4
Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate
(Prophetic)
[0247] Preparation of PLGA-rapamycin conjugate:
[0248] PLGA polymer with acid end group (7525 DLG1A, acid number
0.46 mmol/g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is
dissolved in 30 mL of dichloromethane (DCM).
N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol, 0.57 g) is added
followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and
4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The
mixture is stirred at rt for 2 days. The mixture is then filtered
to remove insoluble dicyclohexylurea. The filtrate is concentrated
to ca. 10 mL in volume and added to 100 mL of isopropyl alcohol
(IPA) to precipitate out the PLGA-rapamycin conjugate. The IPA
layer is removed and the polymer is then washed with 50 mL of IPA
and 50 mL of methyl t-butyl ether (MTBE). The polymer is then dried
under vacuum at 35 C for 2 days to give PLGA-rapamycin as a white
solid (ca. 6.5 g).
[0249] Preparation of nanocarrier containing PLGA-rapamycin
conjugate and ovalbumin peptide (323-339):
[0250] Nanocarrier containing PLGA-rapamycin is prepared according
to the procedure described in Example 1 as follows:
[0251] Solutions for nanocarrier formation are prepared as
follows:
[0252] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution is prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature. Solution 2: PLGA-rapamycin @ 100 mg/mL in
methylene chloride. The solution is prepared by dissolving
PLGA-rapamycin in pure methylene chloride. Solution 3: PLA-PEG @
100 mg/mL in methylene chloride. The solution is prepared by
dissolving PLA-PEG in pure methylene chloride. Solution 4:
Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
[0253] A primary water-in-oil emulsion is prepared first. W1/O1 is
prepared by combining solution 1 (0.2 mL), solution 2 (0.75 mL),
and solution 3 (0.25 mL) in a small pressure tube and sonicating at
50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
A secondary emulsion (W1/O1/W2) is then prepared by combining
solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexing for
10 s, and sonicating at 30% amplitude for 60 seconds using the
Branson Digital Sonifier 250. The W1/O1/W2 emulsion is added to a
beaker containing 70 mM pH 8 phosphate buffer solution (30 mL) and
stirred at room temperature for 2 hours to allow the methylene
chloride to evaporate and for the nanocarriers to form. A portion
of the nanocarriers is washed by transferring the nanocarrier
suspension to a centrifuge tube and centrifuging at 75,600.times.g
and 4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure is repeated, and the pellet is re-suspended in phosphate
buffered saline for a final nanocarrier dispersion of about 10
mg/mL.
Example 5
Polymeric Nanocarrier Containing Polyamino Acid-Lovastatin
Conjugate (Prophetic)
[0254] Preparation of poly-L-glutamic acid-lovastatin conjugate:
Poly-L-glutamic acid sodium salt (5 g) (Alamanda Polymers, 30 kd
MW, 200 L-glutamic acid repeating unit) is dissolved in de-ionized
water (30 mL) and the solution is cooled with ice water. Dilute HCl
solution (1M) is added with vigorous stirring while maintaining
temperature below 10.degree. C. until pH of the solution is about
2.5. The reaction mixture is warmed to room temperature and stirred
for 1 h. The suspension containing insoluble poly-L-glutamic acid
is centrifuged to remove the supernatant (aq. phase). The solid is
then pellet washed with de-ionized water until the wash solution is
at pH>3. The wet solid poly-L-glutamic acid is then lyophilized
to a constant weight as a white powder.
[0255] The poly-L-glutamic acid (2.0 g) is suspended in anhydrous
N,N-diemthylformamide (DMF) (20 mL) with lovastatin (80 mg, 0.2
mmol) and DMAP (2 mmol, 0.24 g). The mixture is stirred at rt for
30 minutes. N,N-diisopropylcarbodiimide (2 mmol, 0.25 g) in 2 mL of
DMF is added dropwise. The reaction mixture is then stirred at rt
overnight. The reaction is cooled with icewater to <5 C and 10%
NaCl solution is added to slowly precipitate out the
poly-L-glutamic acid-lovastatin conjugate. The precipitate is
recovered by centrifuge. The wet solid is then resuspended in water
and the pH of the solution is adjusted to pH 7 with 1M NaHCO3
solution. The resulting solution is stirred for 1 h at rt and
filtered through a 0.2 micron filter to remove impurities. The
filtrate is cooled to <5.degree. C. with ice water and HCl (1N)
is added slowly with vigorous stirring until the pH of the solution
to pH 3. After 30 min of stirring, the precipitate solid is
recovered by centrifuge. The solid is the pellet washed twice with
DI water and then lyophilized to give poly-L-glutamic
acid-lovastatin conjugate (ca. 1.5 g).
[0256] Preparation of nanocarrier containing poly-L-glutamic
acid-lovastatin conjugate and ovalbumin peptide (323-339):
[0257] Nanocarrier containing poly-L-glutamic acid-lovastatin is
prepared according to the procedure described in Example 1 as
follows:
[0258] Solutions for nanocarrier formation are prepared as
follows:
[0259] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in
1.times.PBS buffer aqueous solution. The solution is prepared by
dissolving ovalbumin peptide in 1.times.PBS buffer, pH 7.4 at room
temperature. Solution 2: poly-L-glutamic acid-lovastatin @ 20 mg/mL
in 1.times.PBS buffer. The solution is prepared by dissolving the
polymer in 1.times.PBS buffer. Solution 3: PLGA @ 100 mg/mL in
methylene chloride. The solution was prepared by dissolving PLGA in
pure methylene chloride. Solution 4: Polyvinyl alcohol @ 50 mg/mL
in 100 mM pH 7.4 phosphate buffer.
[0260] A primary water-in-oil emulsion is prepared first. W1/O1 is
prepared by combining solution 1 (0.2 mL), solution 2 (0.75 mL),
and solution 3 (0.25 mL) in a small pressure tube and sonicating at
50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
A secondary emulsion (W1/O1/W2) is then prepared by combining
solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexing for
10 s, and sonicating at 30% amplitude for 60 seconds using the
Branson Digital Sonifier 250. The W1/O1/W2 emulsion is added to a
beaker containing 70 mM pH 7.4 phosphate buffer solution (30 mL)
and stirred at room temperature for 2 hours to allow the methylene
chloride to evaporate and for the nanocarriers to form. A portion
of the nanocarriers is washed by transferring the nanocarrier
suspension to a centrifuge tube and centrifuging at 75,600.times.g
and 4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure is repeated, and the pellet is re-suspended in phosphate
buffered saline for a final nanocarrier dispersion of about 10
mg/mL.
Example 6
Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin
(Prophetic)
[0261] Preparation of HS-PEG-rapamycin:
[0262] A solution of PEG acid disulfide (1.0 eq), rapamycin
(2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred
at rt overnight. The insoluble dicyclohexylurea is removed by
filtration and the filtrate is added to isopropyl alcohol (IPA) to
precipitate out the PEG-disulfide-di-rapamycin ester and washed
with IPA and dried. The polymer is then treated with
tris(2-carboxyethyl)phosphine hydrochloride in DMF to reduce the
PEG disulfide to thiol PEG rapamycin ester (HS-PEG-rapamycin). The
resulting polymer is recovered by precipitation from IPA and dried
as previously described and analyzed by H NMR and GPC.
[0263] Formation of Gold NCs (AuNCs):
[0264] An aq. solution of 500 mL of 1 mM HAuC14 is heated to reflux
for 10 min with vigorous stirring in a 1 L round-bottom flask
equipped with a condenser. A solution of 50 mL of 40 mM of
trisodium citrate is then rapidly added to the stirring solution.
The resulting deep wine red solution is kept at reflux for 25-30
min and the heat is withdrawn and the solution is cooled to room
temperature. The solution is then filtered through a 0.8 .mu.m
membrane filter to give the AuNCs solution. The AuNCs are
characterized using visible spectroscopy and transmission electron
microscopy. The AuNCs are ca. 20 nm diameter capped by citrate with
peak absorption at 520 nm.
[0265] AuNCs conjugate with HS-PEG-rapamycin:
[0266] A solution of 150 .mu.l of HS-PEG-rapamycin (10 .mu.M in 10
mM pH 9.0 carbonate buffer) is added to 1 mL of 20 nm diameter
citrate-capped gold nanocarriers (1.16 nM) to produce a molar ratio
of thiol to gold of 2500:1. The mixture is stirred at room
temperature under argon for 1 hour to allow complete exchange of
thiol with citrate on the gold nanocarriers. The AuNCs with
PEG-rapamycin on the surface is then purified by centrifuge at
12,000 g for 30 minutes. The supernatant is decanted and the pellet
containing AuNC--S-PEG-rapamycin is then pellet washed with
1.times.PBS buffer. The purified Gold-PEG-rapamycin nanocarriers
are then resuspend in suitable buffer for further analysis and
bioassays.
Example 7
Evalutaing Tolerogenic Immune Response to Antigen In vivo
(Prophetic)
[0267] A composition of the invention is dissolved in
phosphate-buffered saline (PBS) or in PBS plus 0.02-0.5% Tween 20
in the case of lipids, and injected into female Lewis rats
intramuscularly in 0.1-0.2 ml containing 500 .mu.g of the
composition. A control group of rats receives 0.1-0.2 ml of
PBS+Tween alone. Within 5 hours after the injection, spleen and
liver are harvested from the rats and single cell suspensions
obtained by macerating tissues through a 40 .mu.m nylon cell
strainer. For the liver, gradient centrifugation steps are
performed to enrich the lymphocyte fraction. Samples are stained in
PBS (1% FCS) with the appropriate dilution of relevant monoclonal
antibodies. Propidum iodide staining cells are excluded from
analysis. Samples are acquired on an LSR2 flow cytometer (BD
Biosciences, USA) and analyzed using FACS Diva software. The
expression of markers CD4, CD25high and FoxP3 is analyzed on iNKT
cells. Also cells can be permebealized and intracellular cytokine
staining can be performed. The presence of CD4+ CD25highFoxP3+
cells suggests an induction of Treg cells. The production of
anti-inflammatory cytokines can suggest tolerizing actions of iNKT
cells. This experiment can also be performed 3 days following the
injection and expansion of iNKT cells and cytokine production can
be measured.
Example 8
Evaluating Tolerogenic Immune Response by Invariant Natural Killer
T-Cell Phenotypic Analysis (Prophetic)
[0268] A composition of the invention is injected subcutaneously
into female Lewis rats. A control group of rats receives 0.1-0.2 ml
of PBS. Nine to ten days after the injection, spleen and lymph
nodes are harvested from the rats and single cell suspensions
obtained by macerating tissues through a 40 .mu.m nylon cell
strainer. Samples are stained in PBS (1% FCS) with the appropriate
dilution of relevant monoclonal antibodies. Propidium iodide
staining cells are excluded from analysis. Samples are acquired on
an LSR2 flow cytometer (BD Biosciences, USA) and analyzed using
FACS Diva software. Invariant natural killer T cells (iNKTs) are
identified by analyzing the expression of CD3, TCR and NK1.1
markers (such as NK1.1 or DX5) and specific T-cell receptor
combinations (invariant Valpha and Vbeta chains). Invariant NKT
cells can also be detected with CD1d tetramers loaded with
.alpha.-galactosylceramide (see, e.g., D. Li et al., Generation and
characterization of CD1d tetramer produced by a lentiviral
expression system. J Immunol Methods. 2008 Jan. 31;
330(1-2):57-63).
Example 9
Evaluating Tolerogenic Immune Response by Assessing Interleukin-10
Production (Prophetic)
[0269] A composition of the invention is injected subcutaneously
into female Lewis rats. A control group of rats receives 0.1-0.2 ml
of PBS. Nine to ten days after the injection, spleen and lymph
nodes are harvested from the rats and single cell suspensions
obtained by macerating tissues through a 40 .mu.m nylon cell
strainer. Samples are stained in PBS (1% FCS) with the appropriate
dilution of relevant monoclonal antibodies for surface antigens. To
stain intracellularly for IL-10, cells are fixed and permeabilized
and IL-10 antibody is added for 30 min. Cells are washed and
propidium iodide staining cells are excluded from analysis. Samples
are acquired on an LSR2 flow cytometer (BD Biosciences, USA) and
analyzed using FACS Diva software. The IL-10 level or the number of
IL-10 producing cells is then compared to a reference level or a
reference number, for example, a level or a number obtained from
the rat before the injection of the composition or a level or
number observed or expected in the control group of rats. An
increase in the level of IL-10 or in the number of IL-10 producing
cells in the rats who received the composition of the invention
suggests an induction of a tolerogenic response.
Example 10
Evaluating iNKT Cell Activation with Synthetic Nanocarriers
Comprising Immunosuppressant
Nanocarriers
[0270] .alpha.-Galactosyl Ceramide (KRN7000) was purchased from
Avanti Polar Lipids, Inc. (700 Industrial Park Drive Alabaster,
Ala. 35007-9105; Catalog number 867000P). PLGA with a
lactide:glycolide ratio of 1:1 and an inherent viscosity of 0.45
dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin
Drive, Birmingham, Ala. 35211; Product Code 5050 DLG 4.5A).
Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD
Chemicals (Product Number 1.41350.1001).
Method
[0271] Solutions were prepared as follows:
[0272] Solution 1: KRN7000 @ 2 mg/mL in dimethylsulfoxide (DMSO).
The solution was prepared by dissolving the dry lipid in pure DMSO.
Solution 2: PLGA @ 100 mg/mL in methylene chloride. The solution
was prepared by dissolving the PLGA in pure methylene chloride.
Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate
buffer.
[0273] A water-in-oil emulsion, (W/O) was prepared by combining
solution 1 (1 mL), solution 2 (1 mL), solution 3 (3 mL) in a small
pressure tube and sonicating at 30% amplitude for 60 seconds using
a Branson Digital Sonifier model 250, with the pressure tube
immersed in an ice water bath. The W/O emulsion was then added to a
beaker containing 70 mM pH 8 phosphate buffer solution (30 mL) and
stirred at room temperature for 2 hours to allow the methylene
chloride to evaporate and for the nanocarriers to form. A portion
of the nanocarriers were washed by transferring the nanocarrier
suspension to a centrifuge tube and centrifuging at 75,600.times.g
at 4.degree. C. for 45 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated, and the pellet was re-suspended in
phosphate buffered saline for a final nanocarrier dispersion of
about 10 mg/mL. Nanocarrier size was determined by dynamic light
scattering. The amount of KRN7000 in the nanocarrier is reported as
the theoretical loading given no loss on processing. The total
dry-nanocarrier mass per mL of suspension (NP concentration), was
determined by a gravimetric method.
TABLE-US-00002 KRN7000 Content Effective (Theoretical % NP
concentration Nanocarrier Diameter (nm) wt/wt) (mg/mL) 212 2.0
9.0
Immunization
[0274] Mice were designated to groups that received PBS alone,
soluble aGC, NPaGC, sol aGC+solRAPA, sol aGC+NP RAPA, NPaGC+solRAPA
or NPaGC+NP RAPA. Mice were injected at 9 am and at 9.30 AM
received BrefeldinA i.v. to prevent release of intracellular
cytokines produced. At 1 pm, mice were sacrificed and the liver was
perfused with PBS and processed to obtain a single cell suspension
enriched for lymphocytes. Cells were stained with cell surface
markers for iNKT cells (agc-loaded CD1d tetramers) and T cells
receptor (TCRb) and activation marker CD69. The cell suspension was
then permeabilized and intracellular cytokine staining was
performed for Th1 cytokine IFN-.gamma., and Th2 cytokines IL-4 and
the anti-inflammatory cytokine IL-10. Cells were acquired on a
FacsCanto flow cytometer and analyzed on by FlowJo.
Results
[0275] iNKT cells from mice that received aGC were activated as
seen by cytokine production and upregulation of CD69 on their
surface. Unexpectedly, it was found that iNKT cells were more
strongly and rapidly activated when they received NP aGC compared
to sol aGC, as seen by rapid downregulation of their TCR. When mice
received RAPA or NP RAPA alone, there was downregulation of CD69,
suggesting the iNKT cells were less active. In addition, a Th2
cytokine skewing with NPaGC was used compared to sol aGC. iNKT
cells produced significantly more IL-4 and IL-10 when activated
with NP aGC compared to sol aGC. The results are shown in FIGS.
2-5.
* * * * *